Therapeutic compositions and methods for malignant tumors with rnai molecules targeted to hsp47 and p21

ABSTRACT

This invention provides compositions and methods for preventing or treating a malignant tumor in a mammal in need thereof, by administering to the mammal a therapeutically effective amount of a composition comprising RNAi molecules, which RNAi molecules can be active in reducing expression of Hsp47, or a combination of RNAi molecules active for Hsp47 and p21.

SEQUENCE LISTING

This application includes a Sequence Listing submitted electronically asan ASCII file created on December xx, 2015, named ND5123767US_SL.txt,which is 100,000 bytes in size, and is hereby incorporated by referencein its entirety.

BACKGROUND OF THE INVENTION

The growth of malignant tumors is related to the extracellular matrix(ECM). The molecular chaperone “heat shock” protein Hsp47 is involved inregulating an ECM gene transcription network.

Hsp47 expression may be activated in breast cancer and other cancers.Reducing Hsp47 can reduce growth of breast cancer cells, and limit tumorgrowth.

Increased expression of Hsp47 may be a factor in poor survival outcomesof breast cancer patients. Hsp47 expression may promote cancerprogression, in part by increasing ECM proteins. See, e.g., Cancer Res,2015, 75(8); 1580-91.

Hsp47 can be highly expressed in pancreatic cancer. Hsp-47 may be auseful specific marker for oral cancer detection.

Hsp47 or a homologous gene sequence thereof is disclosed as, forexample, GenBank accession No. AB010273 (human), X60676 (mouse), orM69246 (rat, gp46).

Agents for suppressing Hsp47 have been disclosed for inhibitingfibrosis. See, e.g., U.S. Pat. No. 8,173,170 B2, and U.S. Pat. No.8,710,209 B2. However, limited information exists concerning the effectof inhibiting Hsp47 in malignant tumor development, progression, andgrowth.

p21 is a cell cycle-regulating protein that is encoded by CDKN1A geneand belongs to the CIP/KIP family. This protein has the function ofinhibiting cell cycle progression at the G1 phase and the G2/M phase byinhibiting the effect of a cyclin-CDK complex through binding to thecomplex. Specifically, the p21 gene undergoes activation by p53, one oftumor suppressor genes. It has been reported that upon activation of p53due to DNA damage or the like, p53 activates p21 so that the cell cycleis arrested at the G1 phase and the G2/M phase.

p21 is overexpressed in a variety of human cancers including prostate,cervical, breast and squamous cell carcinomas and, in many cases, p21upregulation correlates positively with tumor grade, invasiveness andaggressiveness. See, e.g., Chang et al., Proc. Natl. Acad. Sci. USA,2000, Vol. 97, No. 8, pp. 4291-96. Also, up-regulation of p21 has beenreported to be associated with tumorigenicity and poor prognosis in manyforms of cancers, including brain, prostate, ovarian, breast, andesophageal cell cancers. See, e.g., Winters et al., Breast CancerResearch, 2003, Vol. 5, No. 6, pp. R242-R249. Also, the disease can beage related diseases, including atherosclerosis, Alzheimer's disease,amyloidosis, and arthritis. See, e.g., Chang et al., Proc. Natl. Acad.Sci. USA, 2000, Vol. 97, No. 8, pp. 4291-96.

There is an urgent need for methods and compositions to developtherapies for patients with malignant tumors, such as siRNA sequences,compounds and structures for inhibition of expression of Hsp47 and p21.

What is needed are methods and compositions for preventing or treatingmalignant tumors. There is a continuing need for RNAi molecules targetedto Hsp47, p21 and other structures and compositions for preventing,treating, or reducing malignant tumors.

BRIEF SUMMARY

This invention relates to the fields of biopharmaceuticals andtherapeutics composed of nucleic acid based molecules. Moreparticularly, this invention relates to methods and compositions fordelivering RNA interference agents for preventing, treating orameliorating the effects of conditions and diseases involving malignanttumors.

This invention relates to the surprising discovery that malignant tumorsize can be reduced in vivo by treatment with siRNA inhibitors of Hsp47,and inhibitors of Hsp47 in combination with inhibitors of p21.

This invention relates to methods and compositions incorporating nucleicacid based therapeutic compounds for use in delivery to various organsfor preventing, treating, or ameliorating conditions and diseases ofmalignant tumor. In some embodiments, this invention providescompositions of RNA interference molecules (RNAi molecules) for genesilencing of various targets related to malignant tumors.

This invention can provide compositions for delivery of therapeuticmolecules, as well as methods of use thereof. Various RNA-based and drugcompositions of this invention can be used in methods for preventing ortreating malignant tumors.

This invention relates to methods and compositions for nucleic acidbased therapeutic compounds against malignant tumors. In someembodiments, this invention provides RNAi molecules, structures andcompositions that can silence expression of Hsp47, as well as Hsp47 andp21. The structures and compositions of this disclosure can be used inpreventing, treating or reducing the size of malignant tumors.

In certain embodiments, this invention provides double-stranded nucleicacid molecules that are RNAi molecules such as siRNAs or shRNAs forsuppressing Hsp47. Embodiments of this invention can also provide RNAimolecules for suppressing p21.

In certain embodiments, the inhibitory nucleic acid molecule can be anantisense nucleic acid molecule, a small interfering RNA (siRNA), or adouble-stranded RNA (dsRNA).

RNAi molecules of this invention can be active for gene silencing, forexample, a dsRNA that is active for gene silencing, a siRNA, amicro-RNA, or a shRNA active for gene silencing, as well as aDNA-directed RNA (ddRNA), a Piwi-interacting RNA (piRNA), and a repeatassociated siRNA (rasiRNA).

In additional embodiments, methods of this invention can decreasetranscription or translation of Hsp47 and/or p21 in malignant tumors.

In particular embodiments, this invention includes methods fordecreasing expression of Hsp47, or for decreasing expression of Hsp47and p21 in a malignant tumor cell, where the cell can be a human cell, aneoplastic cell, a cell in vivo, or a cell in vitro.

Embodiments of this invention can also provide methods for treating asubject having a neoplasm, where neoplasm cancer cells display aberrantHsp47 expression levels. Methods can involve administering to thesubject an effective amount of an inhibitory nucleic acid molecule,where the inhibitory nucleic acid molecule reduces Hsp47 expression, orwhere a combination of RNAi molecules reduce expression of Hsp47 andp21, thereby treating the neoplasm. In some embodiments, methods of thisinvention can decrease the size of a neoplasm, relative to the size ofthe neoplasm prior to treatment or without treatment.

In various embodiments, an inhibitory nucleic acid molecule can bedelivered in a liposome, a polymer, a microsphere, a nanoparticle, agene therapy vector, or a naked DNA vector.

In further aspects, this invention features methods for treating asubject, e.g. a human patient, having a neoplasm in which the neoplasmcancer cells express Hsp47. In certain embodiments, the methods caninclude administering to the subject an effective amount of inhibitorynucleic acid molecules, where the inhibitory nucleic acid molecules areantisense nucleic acid molecules, or RNAi molecules, or a combinationthereof, which inhibit expression of an Hsp47 polypeptide, or whichinhibit expression of both an Hsp47 polypeptide and a p21 polypeptide.

In particular embodiments, a cell of the neoplasm overexpresses Hsp47.

In certain embodiments, the neoplasm can be a malignant tumor, or lungcancer, or pancreatic cancer.

Embodiments of this invention can provide pharmaceutical compositionsfor treating malignant tumor, the composition comprising nanoparticlesencapsulating RNAi molecules, wherein the RNAi molecules are targeted toHsp47. In some embodiments, this invention includes methods fordistributing an active agent to a subject for treating malignant tumor,the method comprising administering to the subject the pharmaceuticalcomposition above.

In further embodiments, this invention includes pharmaceuticalcompositions for treating malignant tumor, the composition comprisingnanoparticles encapsulating RNAi molecules, wherein a portion of theRNAi molecules are targeted to Hsp47 and a portion of the RNAi moleculesare targeted to p21.

This invention further contemplates methods for preventing, treating orameliorating one or more symptoms of a malignant tumor in a mammal inneed thereof, the method comprising administering to the mammal atherapeutically effective amount of a composition comprising RNAimolecules active in reducing expression of Hsp47.

In further aspects, this invention includes methods for preventing,treating or ameliorating one or more symptoms of a malignant tumor in amammal in need thereof, the method comprising administering to themammal a therapeutically effective amount of a composition comprisingRNAi molecules, wherein a portion of the RNAi molecules are active inreducing expression of Hsp47 and a portion of the RNAi molecules areactive in reducing expression of p21.

In certain aspects, this invention includes methods for reducing thegrowth rate or proliferation of cancer stem cells in a mammal in needthereof, the method comprising administering to the mammal atherapeutically effective amount of a composition comprising RNAimolecules, wherein a portion of the RNAi molecules are active inreducing expression of Hsp47 and a portion of the RNAi molecules areactive in reducing expression of p21.

Additional embodiments of this invention can provide compositions foruse in distributing an active agent for treating a malignant tumor in asubject, wherein the composition comprises liposome nanoparticles. Thecompositions can be used in methods for distributing an active agent toan organ of a subject for treating malignant tumor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of an in vivo study of a pancreatic cancermodel that was performed to test tumor growth inhibition using Hsp47 asa single target. As shown in FIG. 1, for the group treated with aformulation containing Hsp47 siRNA (2M), the tumors grew slowly, if atall, during the course of the study. There was no body weight lossexamined. To the contrary, the tumors of the vehicle control groupdoubled in only 22 days. In conclusion, the Hsp47 siRNA was observed tocompletely suppress tumor growth at dosages of 0.75 mpk, showing thatthe formulation containing the Hsp47 siRNA was a potent anticancertherapeutic.

FIG. 2 shows the gene expressions of Hsp47, collagen I and collagen IVin human cancer cell lines.

FIG. 3 shows an untreated sample for a method for suppressingproliferation of SW480 colon cancer cells with a siRNA targeted toHsp47.

FIG. 4 shows the effect of a negative siRNA at 10 nM in a method forsuppressing proliferation of SW480 colon cancer cells with a siRNAtargeted to Hsp47.

FIG. 5 shows the effect of a negative siRNA at 50 nM in a method forsuppressing proliferation of SW480 colon cancer cells with a siRNAtargeted to Hsp47.

FIG. 6 shows the effect of a negative siRNA at 100 nM in a method forsuppressing proliferation of SW480 colon cancer cells with a siRNAtargeted to Hsp47.

FIG. 7 shows the effect of an active siRNA at 10 nM in a method forsuppressing proliferation of SW480 colon cancer cells with a siRNAtargeted to Hsp47.

FIG. 8 shows the effect of an active siRNA at 50 nM in a method forsuppressing proliferation of SW480 colon cancer cells with a siRNAtargeted to Hsp47.

FIG. 9 shows the effect of an active siRNA at 100 nM in a method forsuppressing proliferation of SW480 colon cancer cells with a siRNAtargeted to Hsp47.

FIG. 10 shows the results of a growth assay in a method for suppressingproliferation of SW480 colon cancer cells with a siRNA targeted toHsp47. The vertical axis is the number of cells×10⁴. Filled circles arethe untreated sample. X markers are the negative siRNA. Triangle markersrepresent the sample treated with an active siRNA targeted to Hsp47.

FIG. 11 shows the results of a dye exclusion assay in a method forsuppressing proliferation of SW480 colon cancer cells with a siRNAtargeted to Hsp47. The vertical axis is the percentage of dead cells.Open circles are the sample treated with an active siRNA targeted toHsp47. X markers are the negative siRNA. Filled circle markers representthe untreated sample.

FIG. 12 shows an untreated sample in a method for suppressingproliferation of HCT116 colon cancer cells with a siRNA targeted toHsp47.

FIG. 13 shows the effect of a negative siRNA at 10 nM in a method forsuppressing proliferation of HCT116 colon cancer cells with a siRNAtargeted to Hsp47.

FIG. 14 shows the effect of an active siRNA at 10 nM in a method forsuppressing proliferation of HCT116 colon cancer cells with a siRNAtargeted to Hsp47.

FIG. 15 shows the results of a growth assay in a method for suppressingproliferation of HCT116 colon cancer cells with a siRNA targeted toHsp47. The vertical axis is the number of cells×10⁴. Filled circles arethe untreated sample. Open circle markers in the rising curve are thenegative siRNA. Open circle markers in the flat curve represent thesample treated with an active siRNA targeted to Hsp47.

FIG. 16 shows the results of a dye exclusion assay in a method forsuppressing proliferation of HCT116 colon cancer cells with a siRNAtargeted to Hsp47. The vertical axis is the percentage of dead cells.Open circles in the rising curve are the sample treated with an activesiRNA targeted to Hsp47. Open circle markers in the flat curve are thenegative siRNA. Filled circle markers represent the untreated sample.

FIG. 17 shows an untreated sample in a method for suppressingproliferation of A549 lung cancer cells with a siRNA targeted to Hsp47.

FIG. 18 shows the effect of a negative siRNA at 10 nM in a method forsuppressing proliferation of A549 lung cancer cells with a siRNAtargeted to Hsp47.

FIG. 19 shows the effect of a negative siRNA at 50 nM in a method forsuppressing proliferation of A549 lung cancer cells with a siRNAtargeted to Hsp47.

FIG. 20 shows the effect of a negative siRNA at 100 nM in a method forsuppressing proliferation of A549 lung cancer cells with a siRNAtargeted to Hsp47.

FIG. 21 shows the effect of an active siRNA at 10 nM in a method forsuppressing proliferation of A549 lung cancer cells with a siRNAtargeted to Hsp47.

FIG. 22 shows the effect of an active siRNA at 50 nM in a method forsuppressing proliferation of A549 lung cancer cells with a siRNAtargeted to Hsp47.

FIG. 23 shows the effect of an active siRNA at 100 nM in a method forsuppressing proliferation of SW480 colon cancer cells with a siRNAtargeted to Hsp47.

FIG. 24 shows the results of a growth assay in a method for suppressingproliferation of A549 lung cancer cells with a siRNA targeted to Hsp47.The vertical axis is the number of cells×10⁴. Filled circles are theuntreated sample. Open circle markers are the negative siRNA. Trianglemarkers represent the sample treated with an active siRNA targeted toHsp47.

FIG. 25 shows the results of a dye exclusion assay in a method forsuppressing proliferation of A549 lung cancer cells with a siRNAtargeted to Hsp47. The vertical axis is the percentage of dead cells.Open circles are the sample treated with an active siRNA targeted toHsp47. X markers are the negative siRNA. Filled circle markers representthe untreated sample.

FIG. 26 shows an untreated sample in a method for suppressingproliferation of HepG2 hepatic cancer cells with a siRNA targeted toHsp47.

FIG. 27 shows the effect of a negative siRNA at 10 nM in a method forsuppressing proliferation of HepG2 hepatic cancer cells with a siRNAtargeted to Hsp47.

FIG. 28 shows the effect of a negative siRNA at 50 nM in a method forsuppressing proliferation of HepG2 hepatic cancer cells with a siRNAtargeted to Hsp47.

FIG. 29 shows the effect of a negative siRNA at 100 nM in a method forsuppressing proliferation of HepG2 hepatic cancer cells with a siRNAtargeted to Hsp47.

FIG. 30 shows the effect of an active siRNA at 10 nM in a method forsuppressing proliferation of HepG2 hepatic cancer cells with a siRNAtargeted to Hsp47.

FIG. 31 shows the effect of an active siRNA at 50 nM in a method forsuppressing proliferation of HepG2 hepatic cancer cells with a siRNAtargeted to Hsp47.

FIG. 32 shows the effect of an active siRNA at 100 nM in a method forsuppressing proliferation of HepG2 hepatic cancer cells with a siRNAtargeted to Hsp47.

FIG. 33 shows the results of a growth assay in a method for suppressingproliferation of HepG2 hepatic cancer cells with a siRNA targeted toHsp47. The vertical axis is the number of cells×10⁴. Filled circles arethe untreated sample. Open circle markers are the negative siRNA. Xmarkers represent the sample treated with an active siRNA targeted toHsp47.

FIG. 34 shows the results of a dye exclusion assay in a method forsuppressing proliferation of HepG2 hepatic cancer cells with a siRNAtargeted to Hsp47. The vertical axis is the percentage of dead cells.Open circles are the sample treated with an active siRNA targeted toHsp47. X markers are the negative siRNA. Open square markers representthe untreated sample.

FIG. 35 shows the results of a method for detecting annexin V and PI incolon cancer cells SW480 transfected with an active Hsp47 siRNA on day 2after transfection of the siRNA.

FIG. 36 shows the results of a method for detecting annexin V and PI incolon cancer cells HCT116 transfected with an active Hsp47 siRNA on day2 after transfection of the siRNA.

FIG. 37 shows the results of a method for detecting expression ofprocaspase-3 and Hsp47 protein in SW480 colon cancer cells transfectedwith an active Hsp47 siRNA.

FIG. 38 shows the results of a method for detecting expression ofprocaspase-3 and Hsp47 protein in HepG2 hepatic cancer cells transfectedwith an active Hsp47 siRNA.

FIG. 39 shows the results of a method for detecting expression ofprocaspase-3 and Hsp47 protein in A549 lung cancer cells transfectedwith an active Hsp47 siRNA.

FIG. 40 shows the results of a method for detecting Caspase-3/7 activityin colon cancer cells SW480 transfected with an Hsp47 siRNA and a p21siRNA. These data show that the use of a combination of an Hsp47 siRNAand a p21 siRNA in colon cancer cells provided unexpectedly advantageousincreases in the levels of Caspases, and that the cells havesurprisingly increased apoptosis.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides methods for utilizing therapeutic compositionsthat decrease the expression of an Hsp47 nucleic acid molecule orpolypeptide for the treatment of a neoplasia in a subject. In certainembodiments, this invention provides methods for utilizing therapeuticcompositions that decrease the expression of an Hsp47 nucleic acidmolecule or polypeptide and a p21 nucleic acid molecule or polypeptidefor the treatment of a neoplasia in a subject.

In additional aspects, embodiments of this invention can provide methodsand compositions for reducing the rate of cancer stem cell growth orproliferation. This invention relates to the surprising effect thatgrowth or proliferation of cancer stem cells can be inhibited in vivo bytreatment with siRNA inhibitors of Hsp47, and inhibitors of Hsp47 incombination with inhibitors of p21.

The therapeutic compositions of this invention can include inhibitorynucleic acid molecules, including RNAi molecules such as siRNAs, shRNAs,and anti sense RNAs.

This invention encompasses RNAi molecules for suppressing DNA encodingHsp47, ribozymes, antisense nucleic acids, DNA/RNA chimericpolynucleotides, and vectors for expressing them, and dominant negativevariants of Hsp47.

In general, after a subject is diagnosed as having a neoplasia, e.g., alung cancer or a pancreatic cancer, a method of treatment involvingsuppression of Hsp47 is selected, or suppression of Hsp47 and p21.

Examples of an agent that suppresses Hsp47 as used herein include a drugthat suppresses Hsp47 production and/or activity, and a drug thatpromotes Hsp47 degradation and/or inactivation. Examples of the drugthat suppresses Hsp47 production include an RNAi molecule, a ribozyme,an antisense nucleic acid, a DNA/RNA chimera polynucleotide for DNAencoding Hsp47, or a vector expressing same.

Examples of an agent that suppresses p21 as used herein include a drugthat suppresses p21 production and/or activity, and a drug that promotesp21 degradation and/or inactivation. Examples of the drug thatsuppresses p21 production include an RNAi molecule, a ribozyme, anantisense nucleic acid, a DNA/RNA chimera polynucleotide for DNAencoding p21, or a vector expressing same.

RNAi Molecules

One of ordinary skill in the art would understand that a reportedsequence may change over time and to incorporate any changes needed inthe nucleic acid molecules herein accordingly.

Embodiments of this invention can provide compositions and methods forgene silencing of Hsp47 expression using small nucleic acid molecules.Additional embodiments of this invention can provide compositions andmethods for gene silencing of Hsp47 expression and p21 expression usingsmall nucleic acid molecules.

RNAi molecules of this invention can be active for gene silencing, forexample, a dsRNA that is active for gene silencing, a siRNA, amicro-RNA, or a shRNA active for gene silencing, as well as aDNA-directed RNA (ddRNA), a Piwi-interacting RNA (piRNA), and a repeatassociated siRNA (rasiRNA). Such molecules are capable of mediating RNAinterference.

The composition and methods disclosed herein can also be used intreating various kinds of malignant tumors in a subject.

The nucleic acid molecules and methods of this invention may be pooled,or used in combination to down regulate the expression of genes thatencode Hsp47, and to down regulate the expression of genes that encodeHsp47 and p21 in concert.

The compositions and methods of this invention can include nucleic acidmolecules, which can modulate or regulate the expression of Hsp47proteins and/or genes encoding the proteins, or which can modulate orregulate the expression of Hsp47 proteins and/or genes encoding theproteins in combination with p21 proteins and/or genes encoding theproteins, as well as proteins and/or genes encoding the proteins thatare associated with the maintenance and/or development of diseases, aswell as conditions or disorders associated with Hsp47, such as malignanttumor.

The compositions and methods of this invention are described withreference to exemplary sequences of Hsp47 and p21. A person of ordinaryskill in the art would understand that various aspects and embodimentsof the invention are directed to any related Hsp47 or p21 genes,sequences, or variants, such as homolog genes and transcript variants,and polymorphisms, including single nucleotide polymorphism (SNP)associated with any Hsp47 or p21 genes.

A RNAi molecule of this invention can be targeted to Hsp47 or p21, andany homologous sequences, for example, using complementary sequences orby incorporating non-canonical base pairs, for example, mismatchesand/or wobble base pairs, that can provide additional target sequences.

In instances where mismatches are identified, non-canonical base pairs,for example, mismatches and/or wobble bases can be used to generatenucleic acid molecules that target more than one gene sequence.

For example, non-canonical base pairs such as UU and CC base pairs canbe used to generate nucleic acid molecules that are capable of targetingsequences for differing targets that share sequence homology. Thus, aRNAi molecule can be targeted to a nucleotide sequence that is conservedbetween homologous genes, and a single RNAi molecule can be used toinhibit expression of more than one gene.

In some aspects, the compositions and methods of this invention includeRNAi molecules that are active against any portion of Hsp47 mRNA. TheRNAi molecule can include a sequence complementary to any mRNA encodinga Hsp47 sequence.

In further aspects, the compositions and methods of this inventioninclude RNAi molecules that are active against any portion of p21 mRNA.The RNAi molecule can include a sequence complementary to any mRNAencoding a p21 sequence.

In some embodiments, a RNAi molecule of this disclosure can haveactivity against Hsp47 RNA, where the RNAi molecule includes a sequencecomplementary to an RNA having a variant Hsp47 encoding sequence, forexample, a mutant Hsp47 gene known in the art to be associated withmalignant tumor.

In further embodiments, a RNAi molecule of this invention can include anucleotide sequence that can mediate silencing of Hsp47 or p21 geneexpression.

As used herein, the RNAi molecule denotes any molecule that causes RNAinterference, including a duplex RNA such as siRNA (small interferingRNA), miRNA (micro RNA), shRNA (short hairpin RNA), ddRNA (DNA-directedRNA), piRNA (Piwi-interacting RNA), or rasiRNA (repeat associated siRNA)and modified forms thereof. These RNAi molecules may be commerciallyavailable or may be designed and prepared based on known sequenceinformation, etc. The antisense nucleic acid includes RNA, DNA, PNA, ora complex thereof. As used herein, the DNA/RNA chimera polynucleotideincludes a double-strand polynucleotide composed of DNA and RNA thatinhibits the expression of a target gene.

In one embodiment, the agents of this invention contain siRNA as atherapeutic agent. An siRNA molecule can have a length from about 10-50or more nucleotides. An siRNA molecule can have a length from about15-45 nucleotides. An siRNA molecule can have a length from about 19-40nucleotides. An siRNA molecule can have a length of from 19-23nucleotides. An siRNA molecule of this invention can mediate RNAiagainst a target mRNA. Commercially available design tools and kits,such as those available from Ambion, Inc. (Austin, Tex.), and theWhitehead Institute of Biomedical Research at MIT (Cambridge, Mass.)allow for the design and production of siRNA.

Methods for Treating Malignant Tumor

Embodiments of this invention can provide RNAi molecules that can beused to down regulate or inhibit the expression of Hsp47 and/or Hsp47proteins, as well as to down regulate or inhibit the expression of p21and/or p21 proteins.

In some embodiments, a RNAi molecule of this invention can be used todown regulate or inhibit the expression of Hsp47 and/or Hsp47 proteinsarising from Hsp47 haplotype polymorphisms that may be associated with adisease or condition such as malignant tumor.

Monitoring of Hsp47 protein or mRNA levels, and/or p21 protein or mRNAlevels, can be used to characterize gene silencing, and to determine theefficacy of compounds and compositions of this invention.

The RNAi molecules of this disclosure can be used individually, or incombination with other siRNAs for modulating the expression of one ormore genes.

The RNAi molecules of this disclosure can be used individually, or incombination, or in conjunction with other known drugs for preventing ortreating diseases, or ameliorating symptoms of conditions or disordersassociated with Hsp47, including malignant tumor.

The RNAi molecules of this invention can be used to modulate or inhibitthe expression of Hsp47 or p21 in a sequence-specific manner.

The RNAi molecules of this disclosure can include a guide strand forwhich a series of contiguous nucleotides are at least partiallycomplementary to a Hsp47 mRNA or a p21 mRNA.

In certain aspects, malignant tumor may be treated by RNA interferenceusing one or more RNAi molecule of this invention.

Treatment of malignant tumor may be characterized in suitable cell-basedmodels, as well as ex vivo or in vivo animal models.

Treatment of malignant tumor may be characterized by determining thelevel of Hsp47 mRNA or the level of Hsp47 protein in cells of affectedtissue, and/or by determining the level of p21 mRNA or the level of p21protein in cells of affected tissue.

Treatment of malignant tumor may be characterized by non-invasivemedical scanning of an affected organ or tissue.

Embodiments of this invention may include methods for preventing,treating, or ameliorating the symptoms of a disease or conditionassociated with Hsp47 in a subject in need thereof.

In certain embodiments, a combination of an Hsp47 siRNA and a p21 siRNAcan provide unexpectedly advantageous increases in cancer cell death. Infurther embodiments, a combination of an Hsp47 siRNA and a p21 siRNA canprovide unexpectedly advantageous decreases in cancer cellproliferation.

In some embodiments, methods for preventing, treating, or amelioratingthe symptoms of malignant tumor in a subject can include administeringto the subject a RNAi molecule of this invention to modulate theexpression of a Hsp47 gene and/or a p21 gene in the subject or organism.

In some embodiments, this invention contemplates methods for downregulating the expression of a Hsp47 gene in a cell or organism, bycontacting the cell or organism with a RNAi molecule of this invention.In certain embodiments, this invention contemplates methods for downregulating the expression of a Hsp47 gene and a p21 gene in a cell ororganism, by contacting the cell or organism with two or more RNAimolecules of this invention.

Inhibitory nucleic acid molecules can be nucleotide oligomers that maybe employed as single-stranded or double-stranded nucleic acid moleculeto decrease gene expression. In one approach, the inhibitory nucleicacid molecule is a double-stranded RNA used for RNA interference(RNAi)-mediated knockdown of gene expression. In one embodiment, adouble-stranded RNA (dsRNA) molecule is made that includes from eight totwenty-five (e.g., 8, 10, 12, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25) consecutive nucleotides of a nucleotide oligomer of the invention.The dsRNA can be two complementary strands of RNA that have duplexed, ora single RNA strand that has self-duplexed (small hairpin (sh)RNA).

In some embodiments, dsRNAs are about 21 or 22 base pairs, but may beshorter or longer, up to about 29 nucleotides. Double stranded RNA canbe made using standard techniques, e.g., chemical synthesis or in vitrotranscription. Kits are available, for example, from Ambion (Austin,Tex.) and Epicentre (Madison, Wis.).

Methods for expressing dsRNA in mammalian cells are described inBrummelkamp et al. Science 296:550-553, 2002; Paddison et al. Genes &Devel. 16:948-958, 2002; Paul et al. Nature Biotechnol. 20:505-508,2002; Sui et al., Proc. Natl. Acad. Sci. USA 99:5515-5520, 2002; Yu etal. Proc. Natl. Acad. Sci. USA 99:6047-6052, 2002; Miyagishi et al.,Nature Biotechnol. 20:497-500, 2002; and Lee et al., Nature Biotechnol.20:500-505 2002, each of which is hereby incorporated by reference.

An inhibitory nucleic acid molecule that “corresponds” to a Hsp47 genecomprises at least a fragment of the double-stranded gene, such thateach strand of the double-stranded inhibitory nucleic acid molecule iscapable of binding to the complementary strand of the target Hsp47 gene.The inhibitory nucleic acid molecule need not have perfectcorrespondence to the reference Hsp47 sequence.

In one embodiment, a siRNA has at least about 85%, 90%, 95%, 96%, 97%,98%, or even 99% sequence identity with the target nucleic acid. Forexample, a 19 base pair duplex having 1-2 base pair mismatch isconsidered useful in the methods of the invention. In other embodiments,the nucleotide sequence of the inhibitory nucleic acid molecule exhibits1, 2, 3, 4, 5 or more mismatches.

The inhibitory nucleic acid molecules provided by the invention are notlimited to siRNAs, but include any nucleic acid molecule sufficient todecrease the expression of a Hsp47 or p21 nucleic acid molecule orpolypeptide. The DNA sequences provided herein may be used, for example,in the discovery and development of therapeutic antisense nucleic acidmolecule to decrease the expression of the encoded protein. Theinvention further provides catalytic RNA molecules or ribozymes. Suchcatalytic RNA molecules can be used to inhibit expression of a targetnucleic acid molecule in vivo. The inclusion of ribozyme sequenceswithin an antisense RNA confers RNA-cleaving activity upon the molecule,thereby increasing the activity of the constructs. The design and use oftarget RNA-specific ribozymes is described in Haseloff et al., Nature334:585-591. 1988, and US 2003/0003469 A1, each of which is incorporatedby reference.

In various embodiments of this invention, the catalytic nucleic acidmolecule is formed in a hammerhead or hairpin motif. Examples of suchhammerhead motifs are described by Rossi et al., Aids Research and HumanRetroviruses, 8:183, 1992. Example of hairpin motifs are described byHampel et al., Biochemistry, 28:4929, 1989, and Hampel et al., NucleicAcids Research, 18: 299, 1990. Those skilled in the art will recognizethat what is needed in an enzymatic nucleic acid molecule is a specificsubstrate binding site that is complementary to one or more of thetarget gene RNA regions, and that it have nucleotide sequences within orsurrounding that substrate binding site which impart an RNA cleavingactivity to the molecule.

Suppression of a target may be determined by the expression or activityof the corresponding protein in cells being suppressed, as compared tocells in which a suppressing agent is not utilized. Expression ofprotein may be evaluated by any known technique; examples thereofinclude an immunoprecipitation method utilizing an antibody, EIA, ELISA,IRA, IRMA, a western blot method, an immunohistochemical method, animmunocytochemical method, a flow cytometry method, varioushybridization methods utilizing a nucleic acid that specificallyhybridizes with a nucleic acid encoding the protein or a unique fragmentthereof, or a transcription product (e.g., mRNA) or splicing product ofsaid nucleic acid, a northern blot method, a Southern blot method, andvarious PCR methods.

The activity of the protein may be evaluated by analyzing a knownactivity of the protein including binding to a protein such as, forexample, Raf-1 (in particular phosphorylated Raf-1) or EGFR (inparticular phosphorylated EGFR) by means of any known method such as forexample an immunoprecipitation method, a western blot method, amassanalysis method, a pull-down method, or a surface plasmon resonance(SPR) method.

In one aspect, the invention features a vector encoding an inhibitorynucleic acid molecule of any of the above aspects. In a particularembodiment, the vector is a retroviral, adenoviral, adeno-associatedviral, or lentiviral vector. In another embodiment, the vector containsa promoter suitable for expression in a mammalian cell.

The amount of active RNA interference inducing ingredient formulated inthe composition of the present invention may be an amount that does notcause an adverse effect exceeding the benefit of administration. Such anamount may be determined by an in vitro test using cultured cells, or atest in a model animal or mammal such as a mouse, a rat, a dog, or apig, etc., and such test methods are known to those skilled in the art.

The amount of active ingredient formulated can vary according to themanner in which the agent or composition is administered. For example,when a plurality of units of the composition is used for oneadministration, the amount of active ingredient to be formulated in oneunit of the composition may be determined by dividing the amount ofactive ingredient necessary for one administration by said plurality ofunits.

This invention also relates to a process for producing an agent orcomposition for suppressing Hsp47 or p21, and the use of a compositionthat suppresses Hsp47, or that suppresses Hsp47 and p21, for reducing orshrinking malignant tumors.

RNA Interference

RNA interference (RNAi) refers to sequence-specific post-transcriptionalgene silencing in animals mediated by short interfering RNAs (siRNAs).See, e.g., Zamore et al., Cell, 2000, Vol. 101, pp. 25-33; Fire et al.,Nature, 1998, Vol. 391, pp. 806811; Sharp, Genes & Development, 1999,Vol. 13, pp. 139-141.

An RNAi response in cells can be triggered by a double stranded RNA(dsRNA), although the mechanism is not yet fully understood. CertaindsRNAs in cells can undergo the action of Dicer enzyme, a ribonucleaseIII enzyme. See, e.g., Zamore et al., Cell, 2000, Vol. 101, pp. 25-33;Hammond et al., Nature, 2000, Vol. 404, pp. 293-296. Dicer can processthe dsRNA into shorter pieces of dsRNA, which are siRNAs.

In general, siRNAs can be from about 21 to about 23 nucleotides inlength and include a base pair duplex region about 19 nucleotides inlength.

RNAi involves an endonuclease complex known as the RNA induced silencingcomplex (RISC). An siRNA has an antisense or guide strand which entersthe RISC complex and mediates cleavage of a single stranded RNA targethaving a sequence complementary to the antisense strand of the siRNAduplex. The other strand of the siRNA is the passenger strand. Cleavageof the target RNA takes place in the middle of the region complementaryto the antisense strand of the siRNA duplex See, e.g., Elbashir et al.,Genes & Development, 2001, Vol. 15, pp. 188-200.

As used herein, the term “sense strand” refers to a nucleotide sequenceof a siRNA molecule that is partially or fully complementary to at leasta portion of a corresponding antisense strand of the siRNA molecule. Thesense strand of a siRNA molecule can include a nucleic acid sequencehaving homology with a target nucleic acid sequence.

As used herein, the term “antisense strand” refers to a nucleotidesequence of a siRNA molecule that is partially or fully complementary toat least a portion of a target nucleic acid sequence. The antisensestrand of a siRNA molecule can include a nucleic acid sequence that iscomplementary to at least a portion of a corresponding sense strand ofthe siRNA molecule.

RNAi molecules can down regulate or knock down gene expression bymediating RNA interference in a sequence-specific manner. See, e.g.,Zamore et al., Cell, 2000, Vol. 101, pp. 25-33; Elbashir et al., Nature,2001, Vol. 411, pp. 494-498; Kreutzer et al., WO2000/044895;Zernicka-Goetz et al., WO2001/36646; Fire et al., WO1999/032619;Plaetinck et al., WO2000/01846; Mello et al., WO2001/029058.

As used herein, the terms “inhibit,” “down-regulate,” or “reduce” withrespect to gene expression means that the expression of the gene, or thelevel of mRNA molecules encoding one or more proteins, or the activityof one or more of the encoded proteins is reduced below that observed inthe absence of a RNAi molecule or siRNA of this invention. For example,the level of expression, level of mRNA, or level of encoded proteinactivity may be reduced by at least 1%, or at least 10%, or at least20%, or at least 50%, or at least 90%, or more from that observed in theabsence of a RNAi molecule or siRNA of this invention.

RNAi molecules can also be used to knock down viral gene expression, andtherefore affect viral replication.

RNAi molecules can be made from separate polynucleotide strands: a sensestrand or passenger strand, and an antisense strand or guide strand. Theguide and passenger strands are at least partially complementary. Theguide strand and passenger strand can form a duplex region having fromabout 15 to about 49 base pairs.

In some embodiments, the duplex region of a siRNA can have 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 base pairs.

In certain embodiments, a RNAi molecule can be active in a RISC complex,with a length of duplex region active for RISC.

In additional embodiments, a RNAi molecule can be active as a Dicersubstrate, to be converted to a RNAi molecule that can be active in aRISC complex.

In some aspects, a RNAi molecule can have complementary guide andpassenger sequence portions at opposing ends of a long molecule, so thatthe molecule can form a duplex region with the complementary sequenceportions, and the strands are linked at one end of the duplex region byeither nucleotide or non-nucleotide linkers. For example, a hairpinarrangement, or a stem and loop arrangement. The linker interactionswith the strands can be covalent bonds or non-covalent interactions.

A RNAi molecule of this disclosure may include a nucleotide,non-nucleotide, or mixed nucleotide/non-nucleotide linker that joins thesense region of the nucleic acid to the antisense region of the nucleicacid. A nucleotide linker can be a linker of 2 nucleotides in length,for example about 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. Thenucleotide linker can be a nucleic acid aptamer. By “aptamer” or“nucleic acid aptamer” as used herein refers to a nucleic acid moleculethat binds specifically to a target molecule wherein the nucleic acidmolecule has sequence that includes a sequence recognized by the targetmolecule in its natural setting. Alternately, an aptamer can be anucleic acid molecule that binds to a target molecule, where the targetmolecule does not naturally bind to a nucleic acid. For example, theaptamer can be used to bind to a ligand-binding domain of a protein,thereby preventing interaction of the naturally occurring ligand withthe protein. See, e.g., Gold et al., Annu Rev Biochem, 1995, Vol. 64,pp. 763-797; Brody et al., J. Biotechnol., 2000, Vol. 74, pp. 5-13;Hermann et al., Science, 2000, Vol. 287, pp. 820-825.

Examples of a non-nucleotide linker include an abasic nucleotide,polyether, polyamine, polyamide, peptide, carbohydrate, lipid,polyhydrocarbon, or other polymeric compounds, for example polyethyleneglycols such as those having from 2 to 100 ethylene glycol units. Someexamples are described in Seela et al., Nucleic Acids Research, 1987,Vol. 15, pp. 3113-3129; Cload et al., J. Am. Chem. Soc., 1991, Vol. 113,pp. 6324-6326; Jaeschke et al., Tetrahedron Lett., 1993, Vol. 34, pp.301; Arnold et al., WO1989/002439; Usman et al., WO1995/006731; Dudyczet al., WO1995/011910, and Ferentz et al., J. Am. Chem. Soc., 1991, Vol.113, pp. 4000-4002.

A RNAi molecule can have one or more overhangs from the duplex region.The overhangs, which are non-base-paired, single strand regions, can befrom one to eight nucleotides in length, or longer. An overhang can be a3′-end overhang, wherein the 3′-end of a strand has a single strandregion of from one to eight nucleotides. An overhang can be a 5′-endoverhang, wherein the 5′-end of a strand has a single strand region offrom one to eight nucleotides.

The overhangs of a RNAi molecule can have the same length, or can bedifferent lengths.

A RNAi molecule can have one or more blunt ends, in which the duplexregion ends with no overhang, and the strands are base paired to the endof the duplex region.

A RNAi molecule of this disclosure can have one or more blunt ends, orcan have one or more overhangs, or can have a combination of a blunt endand an overhang end.

A 5′-end of a strand of a RNAi molecule may be in a blunt end, or can bein an overhang. A 3′-end of a strand of a RNAi molecule may be in ablunt end, or can be in an overhang.

A 5′-end of a strand of a RNAi molecule may be in a blunt end, while the3′-end is in an overhang. A 3′-end of a strand of a RNAi molecule may bein a blunt end, while the 5′-end is in an overhang.

In some embodiments, both ends of a RNAi molecule are blunt ends.

In additional embodiments, both ends of a RNAi molecule have anoverhang.

The overhangs at the 5′- and 3′-ends may be of different lengths.

In certain embodiments, a RNAi molecule may have a blunt end where the5′-end of the antisense strand and the 3′-end of the sense strand do nothave any overhanging nucleotides.

In further embodiments, a RNAi molecule may have a blunt end where the3′-end of the antisense strand and the 5′-end of the sense strand do nothave any overhanging nucleotides.

A RNAi molecule may have mismatches in base pairing in the duplexregion.

Any nucleotide in an overhang of a RNAi molecule can be adeoxyribonucleotide, or a ribonucleotide.

One or more deoxyribonucleotides may be at the 5′-end, where the 3′-endof the other strand of the RNAi molecule may not have an overhang, ormay not have a deoxyribonucleotide overhang.

One or more deoxyribonucleotides may be at the 3′-end, where the 5′-endof the other strand of the RNAi molecule may not have an overhang, ormay not have a deoxyribonucleotide overhang.

In some embodiments, one or more, or all of the overhang nucleotides ofa RNAi molecule may be 2′-deoxyribonucleotides.

Dicer Substrate RNAi Molecules

In some aspects, a RNAi molecule can be of a length suitable as a Dicersubstrate, which can be processed to produce a RISC active RNAimolecule. See, e.g., Rossi et al., US2005/0244858.

A Dicer substrate dsRNA can be of a length sufficient such that it isprocessed by Dicer to produce an active RNAi molecule, and may furtherinclude one or more of the following properties: (i) the Dicer substratedsRNA can be asymmetric, for example, having a 3′ overhang on theantisense strand, and (ii) the Dicer substrate dsRNA can have a modified3′ end on the sense strand to direct orientation of Dicer binding andprocessing of the dsRNA to an active RNAi molecule.

RNAi Molecules for p21

Examples of RNAi molecules of this invention targeted to p21 mRNA areshown in Table 1.

TABLE 1 RNAi molecule sequences for p21 SEQ SENSE STRAND SEQANTISENSE STRAND Ref  ID (5′-->3′) ID (5′-->3′) Pos NOSEQ ID NOS: 1 to 28 NO SEQ ID NOS: 29 to 56 2085  1CUUAGUGACUUUACUUGUAmUmU 29 UACAAGUAAAGUCACUAAGmUmU  500  2CAGACCAGCAUGACAGAUUmUmU 30 AAUCUGUCAUGCUGGUCUGmUmU  540  3UGAUCUUCUCCAAGAGGAAmUmU 31 UUCCUCUUGGAGAAGAUCAmUmU 1706  4GUUCAUUGCACUUUGAUUAmUmU 32 UAAUCAAAGUGCAAUGAACmUmU 1709  5CAUUGCACUUUGAUUAGCAmUmU 33 UGCUAAUCAAAGUGCAAUGmUmU  210  6AGCGAUGGAACUUCGACUUmUmU 34 AAGUCGAAGUUCCAUCGCUmUmU  211  7GCGAUGGAACUUCGACUUUmUmU 35 AAAGUCGAAGUUCCAUCGCmUmU 1473  8GGGAAGGGACACACAAGAAmUmU 36 UUCUUGUGUGUCCCUUCCCmUmU 1507  9UCUACCUCAGGCAGCUCAAmUmU 37 UUGAGCUGCCUGAGGUAGAmUmU 2067 10GGUGCUCAAUAAAUGAUUCmUmU 38 GAAUCAUUUAUUGAGCACCmUmU 1063 11CAUCAUCAAAAACUUUGGAmUmU 39 UCCAAAGUUUUUGAUGAUGmUmU 1735 12AAGGAGUCAGACAUUUUAAmUmU 40 UUAAAAUGUCUGACUCCUUmUmU  783 13GUGCUGGGCAUUUUUAUUUmUmU 41 AAAUAAAAAUGCCCAGCACmUmU  869 14GCCGGCUUCAUGCCAGCUAmUmU 42 UAGCUGGCAUGAAGCCGGCmUmU 1060 15GGGCAUCAUCAAAAACUUUmUmU 43 AAAGUUUUUGAUGAUGCCCmUmU 1492 16GAAGGGCACCCUAGUUCUAmUmU 44 UAGAACUAGGGUGCCCUUCmUmU 1704 17CAGUUCAUUGCACUUUGAUmUmU 45 AUCAAAGUGCAAUGAACUGmUmU 1733 18ACAAGGAGUCAGACAUUUUmUmU 46 AAAAUGUCUGACUCCUUGUmUmU 1847 19UGGAGGCACUGAAGUGCUUmUmU 47 AAGCACUUCAGUGCCUCCAmUmU 2000 20GCAGGGACCACACCCUGUAmUmU 48 UACAGGGUGUGGUCCCUGCmUmU 2014 21CUGUACUGUUCUGUGUCUUmUmU 49 AAGACACAGAACAGUACAGmUmU  677 22UUAAACACCUCCUCAUGUAmUmU 50 UACAUGAGGAGGUGUUUAAmUmU  475 23AGACUCUCAGGGUCGAAAAmUmU 51 UUUUCGACCCUGAGAGUCUmUmU  508 24CAUGACAGAUUUCUACCACmUmU 52 GUGGUAGAAAUCUGUCAUGmUmU  514 25AGAUUUCUACCACUCCAAAmUmU 53 UUUGGAGUGGUAGAAAUCUmUmU  549 26CCAAGAGGAAGCCCUAAUCmUmU 54 GAUUAGGGCUUCCUCUUGGmUmU  382 27GACAGCAGAGGAAGACCAUmUmU 55 AUGGUCUUCCUCUGCUGUCmUmU 2042 28CUCCCACAAUGCUGAAUAUmUmU 56 AUAUUCAGCAUUGUGGGAGmUmU

Key for Table 1: Upper case A, G, C and U referred to for ribo-A,ribo-G, ribo-C and ribo-U respectively. The lower case letters a, g, c,t represent 2′-deoxy-A, 2′-deoxy-G, 2′-deoxy-C and thymidinerespectively. mU is 2′-methoxy-U.

Examples of RNAi molecules of this invention targeted to p21 mRNA areshown in Table 2.

TABLE 2 RNAi molecule sequences for p21 SEQ SENSE STRAND SEQANTISENSE STRAND Ref  ID (5′-->3′) ID (5′-->3′) Pos NOSEQ ID NOS: 57 to 70 NO SEQ ID NOS: 71 to 84 1735′ 57AAGGAGUCAGACAUUUUAANN 71 UUAAAAUGUCUGACUCCUUNN    1 58AAGGAGUCAGACAUUUUAAUU 72 UUAaAaUgUCUGACUCCUUUU    2 59AAGGAGUCAGACAUUUUAAUU 73 UUAaAaUgUCUGACUCCUUUU    3 60AAGGAGUCAGACAUUUUAAUU 74 UUAaAaUgUCUGACUCCUUUU    4 61AAGGAGUCAGACAUUUUAAUU 75 UUAaAaUgUCUGACUCCUUUU    5 62AAGGAGUCAGACAUUUUAAUU 76 UUaaaaugUCUGACUCCUUUU    6 63AAGGAGUCAGACAUUUUAAUU 77 UUAAaaugUCUGACUCCUUUU    7 64AAGGAGUCAGACAUUUUAAUU 78 uUaAaAuGUCUGACUCCUUUU    8 65AAGGAGUCAGACAUUUUAAUU 79 UUaAaAuGUCUGACUCCUUUU    9 66AAGGAGUCAGACAUUUUAAUU 80 UUAaAaUgUCUGACUCCUUUU   10 67AAGGAGUCAGACAUUUUAAUU 81 UUAAAAUGUCUGACUCCUUUU   11 68AAGGAGUCAGACAUUUUAAUU 82 UUAAAAUGUCUGACUCCUUUU   12 69AAGGAGUCAGACAUUUUAAUU 83 UUAAAAUGUCUGACUCCUUUU   13 70AAGGAGUCAGACAUUUUAAUU 84 UUAAAAUGUCUGACUCCUUUU

Key for Table 2: Upper case A, G, C and U refer to ribo-A, ribo-G,ribo-C and ribo-U, respectively. The lower case letters a, u, g, c, trefer to 2′-deoxy-A, 2′-deoxy-U, 2′-deoxy-G, 2′-deoxy-C, anddeoxythymidine (dT=T=t) respectively. Underlining refers to2′-OMe-substituted, e.g., U. N is A, C, G, U, U, a, c, g, u, t, or amodified, inverted, or chemically modified nucleotide.

As used herein, the RNAi molecule denotes any molecule that causes RNAinterference, including a duplex RNA such as siRNA (small interferingRNA), miRNA (micro RNA), shRNA (short hairpin RNA), ddRNA (DNA-directedRNA), piRNA (Piwi-interacting RNA), or rasiRNA (repeat associated siRNA)and modified forms thereof. These RNAi molecules may be commerciallyavailable or may be designed and prepared based on known sequenceinformation, etc. The antisense nucleic acid includes RNA, DNA, PNA, ora complex thereof. As used herein, the DNA/RNA chimera polynucleotideincludes a double-strand polynucleotide composed of DNA and RNA thatinhibits the expression of a target gene.

In one embodiment, the agents of this invention contain siRNA as atherapeutic agent. An siRNA molecule can have a length from about 10-50or more nucleotides. An siRNA molecule can have a length from about15-45 nucleotides. An siRNA molecule can have a length from about 19-40nucleotides. An siRNA molecule can have a length of from 19-23nucleotides. An siRNA molecule of this invention can mediate RNAiagainst a target mRNA. Commercially available design tools and kits,such as those available from Ambion, Inc. (Austin, Tex.), and theWhitehead Institute of Biomedical Research at MIT (Cambridge, Mass.)allow for the design and production of siRNA.

p21 is present in various animals including humans. Sequence informationfor human CDKN1A (p21) is found at: NM_000389.4, NM_078467.2,NM_001291549.1, NM_001220778.1, NM_001220777.1 (NP_001207707.1,NP_001278478.1, NP_001207706.1, NP_510867.1, NP_000380.1).

The nucleic acid sequence of an example target p21 mRNA is disclosed inGenBank accession number NM_000389.4 (CDKN1A), which is 2175 nucleotidesin length.

RNAi Molecules Targeted for Hsp47

In some embodiments, this invention can provide a range of RNAimolecules and compositions for modulating expression of heat shockprotein 47 (Hsp47), a collagen-specific molecular chaperone forintracellular transport and maturation.

Some examples of siRNAs for Hsp47 are given in U.S. Pat. No. 8,710,209,which is hereby incorporated by reference in its entirety for allpurposes.

Hsp47 or a homologous gene sequence thereof is disclosed as, forexample, GenBank accession No. AB010273 (human), X60676 (mouse), orM69246 (rat, gp46).

Agents for suppressing Hsp47 have been disclosed for inhibitingfibrosis. See, e.g., U.S. Pat. No. 8,173,170 B2, which is herebyincorporated by reference in its entirety for all purposes. However,limited information exists concerning the effect of inhibiting Hsp47 inmalignant tumor development, progression, and growth.

In some embodiments, each strand of a siRNA molecule of this inventioncan be from 15 to 60 nucleotides in length, or from 15 to 40 nucleotidesin length, or from 19 to 25 nucleotides in length.

In certain embodiments, this invention provides a pharmaceuticalcomposition containing RNAi molecules for treating malignant tumor thatare RNAi molecules targeted to Hsp47.

Examples of RNAi molecules of this disclosure targeted to Hsp47 mRNA areshown in Table 3.

TABLE 3 RNAi molecule sequences for Hsp47 SEQ SENSE STRAND SEQANTISENSE STRAND Ref  ID (5′-->3′) ID (5′-->3′) Pos NOSEQ ID NOS: 85 to 105 NO SEQ ID NOS: 106 to 126 mouse  85CGAGAACAGUUUGUACAAGUU 106 CUUGUACAAACUGUUCUCGUU  86CAGGCCUCUACAACUACUATT 107 UAGUAGUUGUAGAGGCCUGTT  87GAGCACUCCAAGAUCAACUUCCG 108 CGCGGAAGUUGAUCUUGGAGUGC CG UCUU  88GGACAGGCCUCUACAACUATT 109 UAGUUGUAGAGGCCUGUCCTT  89GAGCACUCCAAGAUCAACUTT 110 AGUUGAUCUUGGAGUGCUCTT  90GAACACUCCAAGAUCAACUTT 111 AGUUGAUCUUGGAGUGUUCTT  91CAGGCCUCUACAACUACUACGAC 112 UCGUCGUAGUAGUUGUAGAGGCC GA UGUU  92GAACACUCCAAGAUCAACUUCCG 113 CUCGGAAGUUGAUCUUGGAGUGU AG UCUU  93GGACAGGCCUCUACAACUACUAC 114 UCGUAGUAGUUGUAGAGGCCUGU GA CCUU  94CAGGCCUCUACAACUACUAdTdT 115 UAGUAGUUGUAGAGGCCUGdTdT dAdAdAdAdA  95CAGGCCUCUACAACUACUA 116 UAGUAGUUGUAGAGGCCUGdTdT  96CAGGCCUCUACAACUACUAdTdT 117 UAGUAGUUGUAGAGGCCUGdTdTdAdAdAdAdAdAdAdAdAdAdAd A  97 dAdAdAdAdACAGGCCUCUACAA 118UAGUAGUUGUAGAGGCCUGdTdT CUACUAdTdT  98 CAGGCCUCUACAACUACUAdTdT 119UAGUAGUUGUAGAGGCCUGdTdT dAdAdAdAdAdAdAdA  99 dAdAdAdAdAdAdAdACAGGCCU 120UAGUAGUUGUAGAGGCCUGdTdT CUACAACUACUAdTdT 100 CAGGCCUCUACAACUACUAdTdT 121UAGUAGUUGUAGAGGCCUGdTdT dAdAdAdAdAdAdAdAdAdAdAd A 101dAdAdAdAdAdAdAdAdAdAdAd 122 UAGUAGUUGUAGAGGCCUGdTdTAdAdAdAdACAGGCCUCUACAAC UACUAdTdT 102 CAGGCCUCUACAACUACUAdTdT 123UAGUAGUUGUAGAGGCCUGdTdT dAdAdAdAdAdAdAdAdAdAdAd AdAdAdAdA 103CAGGCCUCUACAACUACUAdTdT 124 UAGUAGUUGUAGAGGCCUGdTdT mouse 104GGACAGGCCUGUACAACUAdTdT 125 UAGUUGUACAGGCCUGUCCdTdT human 105GGACAGGCCUCUACAACUAdTdT 126 UAGUUGUAGAGGCCUGUCCdTdT

Key for Table 3: Upper case A, G, C and U referred to for ribo-A,ribo-G, ribo-C and ribo-U respectively. Lower case d represents “deoxy.”

Additional examples of RNAi molecules of this disclosure targeted toHsp47 mRNA are shown in Table 4.

TABLE 4 RNAi molecule sequences and control for Hsp47 SEQ ID NO SEQUENCE127 3′-C3-25rU-25rC-25rC-25rU-25rU-rC- SENSErA-rA-rC-rU-rA-rG-rA-rA-rC-rC-rU- 45-M rC-rA-idAB-5′ 1283′-C3-C3-mU-rG-mA-rG-mG-rU-mU-rC- ANTISENSEmU-rA-rG-mU-25rU-mG-rA-mA-rG-mG- 45-M rA-5′ 1293′-C3-rA-rG-mU-rG-rG-rG-mU-rA-mC- SENSE rA-25rC-rA-rG-rA-rG-mU-rC-rC-rU-51_M idAB-5′ 130 3′-C3-C3-rA-rG-rG-rA-mC-rU-rC-rU- ANTISENSEmG-rU-rG-mU-rA-25rC-rC-mC-rA-rC- 51_M mU-5′ 1315′-idAB-rG-rA-rG-rA-rC-rA-rC-rA- SENSE rU-rG-rG-rG-rU-rG-25rC-25rU-25rA-2_M 25rU-25rA-C3-P-3′ 132 5′-mU-rA-mU-rA-mG-rC-25rA-rC-mC- ANTISENSErC-mA-rU-mG-rU-mG-rU-mC-rU-mC-C3- 2_M C3-3′ 1335′-idAB-rC-mU-mU-rA-mC-rG-mC-mU- SENSE 25rG-rA-rG-mU-rA-mC-mU-rU-mC-rG-Negative  rU-C3-3′ control 134 5′-rA-mC-rG-rA-rA-25rG-mU-rA-rC-ANTISENSE rU-mC-rA-rG-rC-rG-mU-rA-rA-rG-C3- Negative  C3-3′ control

Key for Table 4: Designations: rXrepresents ribonucleotides, mXrepresents 2′-O-Methyl ribonucleotides, 25rX represents ribonucleotideswith 2′-5′ linkages, C3 represents a 1,3-propanediol spacer, idABrepresents inverted 1,2-dideoxy-D-Ribose, P represents a phosphate groupon the 3′-terminus.

Methods of Use of RNAi Molecules

The nucleic acid molecules and RNAi molecules of this invention may bedelivered to a cell or tissue by direct application of the molecules, orwith the molecules combined with a carrier or a diluent.

The nucleic acid molecules and RNAi molecules of this invention can bedelivered or administered to a cell, tissue, organ, or subject by directapplication of the molecules with a carrier or diluent, or any otherdelivery vehicle that acts to assist, promote or facilitate entry into acell, for example, viral sequences, viral material, or lipid or liposomeformulations.

The nucleic acid molecules and RNAi molecules of this invention can becomplexed with cationic lipids, packaged within liposomes, or otherwisedelivered to target cells or tissues. The nucleic acid or nucleic acidcomplexes can be locally administered to relevant tissues ex vivo, or invivo through direct dermal application, transdermal application, orinjection.

Delivery systems may include, for example, aqueous and nonaqueous gels,creams, emulsions, microemulsions, liposomes, ointments, aqueous andnonaqueous solutions, lotions, aerosols, hydrocarbon bases and powders,and can contain excipients such as solubilizers and permeationenhancers.

A inhibitory nucleic acid molecule or composition of this invention maybe administered within a pharmaceutically-acceptable diluents, carrier,or excipient, in unit dosage form. Conventional pharmaceutical practicemay be employed to provide suitable formulations or compositions toadminister the compounds to patients suffering from a disease that iscaused by excessive cell proliferation. Administration may begin beforethe patient is symptomatic. Any appropriate route of administration maybe employed, for example, administration may be parenteral, intravenous,intraarterial, subcutaneous, intratumoral, intramuscular, intracranial,intraorbital, ophthalmic, intraventricular, intrahepatic, intracapsular,intrathecal, intracistemal, intraperitoneal, intranasal, aerosol,suppository, or oral administration. For example, therapeuticformulations may be in the form of liquid solutions or suspensions; fororal administration, formulations may be in the form of tablets orcapsules; and for intranasal formulations, in the form of powders, nasaldrops, or aerosols.

Compositions and methods of this disclosure can include an expressionvector that includes a nucleic acid sequence encoding at least one RNAimolecule of this invention in a manner that allows expression of thenucleic acid molecule.

The nucleic acid molecules and RNAi molecules of this invention can beexpressed from transcription units inserted into DNA or RNA vectors.Recombinant vectors can be DNA plasmids or viral vectors. Viral vectorscan be used that provide for transient expression of nucleic acidmolecules.

For example, the vector may contain sequences encoding both strands of aRNAi molecule of a duplex, or a single nucleic acid molecule that isself-complementary and thus forms a RNAi molecule. An expression vectormay include a nucleic acid sequence encoding two or more nucleic acidmolecules.

A nucleic acid molecule may be expressed within cells from eukaryoticpromoters. Those skilled in the art realize that any nucleic acid can beexpressed in eukaryotic cells from the appropriate DNA/RNA vector.

In some aspects, a viral construct can be used to introduce anexpression construct into a cell, for transcription of a dsRNA constructencoded by the expression construct.

Lipid formulations can be administered to animals by intravenous,intramuscular, or intraperitoneal injection, or orally or by inhalationor other methods as are known in the art.

Pharmaceutically acceptable formulations for administeringoligonucleotides are known and can be used.

In one embodiment of the above method, the inhibitory nucleic acidmolecule is administered at a dosage of about 5 to 500 mg/m²/day, e.g.,5, 25, 50, 100, 125, 150, 175, 200, 225, 250, 275, or 300 mg/m²/day.

In some embodiments, the inhibitory nucleic acid molecules of thisinvention are administered systemically in dosages from about 1 to 100mg/kg, e.g., 1, 5, 10, 20, 25, 50, 75, or 100 mg/kg.

In further embodiments, the dosage can range from about 25 to 500mg/m²/day.

Methods known in the art for making formulations are found, for example,in “Remington: The Science and Practice of Pharmacy” Ed. A. R. Gennaro,Lippincourt Williams & Wilkins, Philadelphia, Pa., 2000.

Formulations for parenteral administration may, for example, containexcipients, sterile water, or saline, polyalkylene glycols such aspolyethylene glycol, oils of vegetable origin, or hydrogenatednapthalenes. Biocompatible, biodegradable lactide polymer,lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylenecopolymers may be used to control the release of the compounds. Otherpotentially useful parenteral delivery systems for inhibitory nucleicacid molecules include ethylene-vinyl acetate copolymer particles,osmotic pumps, implantable infusion systems, and liposomes. Formulationsfor inhalation may contain excipients, for example, lactose, or may beaqueous solutions containing, for example, polyoxyethylene-9-laurylether, glycocholate and deoxycholate, or may be oily solutions foradministration in the form of nasal drops, or as a gel.

The formulations can be administered to human patients intherapeutically effective amounts (e.g., amounts which prevent,eliminate, or reduce a pathological condition) to provide therapy for aneoplastic disease or condition. The preferred dosage of a nucleotideoligomer of the invention can depend on such variables as the type andextent of the disorder, the overall health status of the particularpatient, the formulation of the compound excipients, and its route ofadministration.

A pharmaceutical composition of this invention can be effective intreating a Hsp47 associated disease. Examples of the diseases include adisease due to abnormal cell proliferation, and a disease presentingHsp47 overexpression.

All of the above methods for reducing malignant tumors may be either anin vitro method or an in vivo method. Dosage may be determined by an invitro test using cultured cells, etc., as is known in the art. Aneffective amount may be an amount that reduces tumor size by at least10%, at least 20%, or at least 30%, or at least 40%, or at least 50%, orat least 60%, or at least 70%, or at least 80%, or at least 90%, up to100% of the tumor size. An effective amount may be an amount thatreduces cancer cell proliferation by at least 10%, at least 20%, or atleast 30%, or at least 40%, or at least 50%, or at least 60%, or atleast 70%, or at least 80%, or at least 90%, or up to 100% as comparedto control.

Examples of the disease due to abnormal cell proliferation includemalignant tumors, hyperplasia, keloid, Cushing's syndrome, primaryaldosteronism, erythroplakia, polycythemia vera, leukoplakia,hyperplastic scar, lichen planus, and lentiginosis.

Examples of the disease due to Hsp47 overexpression include malignanttumor.

Examples of cancer include sarcomas such as fibrosarcoma, malignantfibrous histiocytoma, liposarcoma, rhabdomyosarcoma, leiomyosarcoma,angiosarcoma, Kaposi's sarcoma, lymphangiosarcoma, synovial sarcoma,chondrosarcoma, and osteosarcoma, carcinomas such as brain tumor, headand neck carcinoma, breast carcinoma, lung carcinoma, esophagealcarcinoma, gastric carcinoma, duodenal carcinoma, appendiceal carcinoma,colon carcinoma, rectal carcinoma, liver carcinoma, pancreaticcarcinoma, gall bladder carcinoma, bile duct carcinoma, anal carcinoma,renal carcinoma, ureteral carcinoma, bladder carcinoma, prostatecarcinoma, testicular carcinoma, uterine carcinoma, ovarian carcinoma,skin carcinoma, leukemia, and malignant lymphoma.

Cancer includes epithelial malignancy and non-epithelial malignancy. Acancer can be present at any site of the body, for example, the brain,head and neck, chest, limbs, lung, heart, thymus, esophagus, stomach,small intestine (duodenum, jejunum, ileum), large intestine (colon,cecum, appendix, rectum), liver, pancreas, gallbladder, kidney, urinaryduct, bladder, prostate, testis, uterus, ovary, skin, striated muscle,smooth muscle, synovial membrane, cartilage, bone, thyroid, adrenalgland, peritoneum, mesentery, bone marrow, blood, vascular system,lymphatic system such as lymph node, lymphatic fluid, etc.

In another embodiment, the cancer includes cancer cells that exhibithormone- or growth factor-independent proliferation. In furtherembodiments, a cancer includes cancer cells exhibiting Hsp47overexpression.

Nanoparticles

Embodiments of this invention can provide liposome nanoparticlecompositions. The ionizable molecules of this invention can be used toform liposome compositions, which can have a bilayer of lipid-likemolecules.

A nanoparticle composition can have one or more of the ionizablemolecules of this invention in a liposomal structure, a bilayerstructure, a micelle, a lamellar structure, or a mixture thereof.

In some embodiments, a composition can include one or more liquidvehicle components. A liquid vehicle suitable for delivery of activeagents of this invention can be a pharmaceutically acceptable liquidvehicle. A liquid vehicle can include an organic solvent, or acombination of water and an organic solvent.

Embodiments of this invention can provide lipid nanoparticles having asize of from 10 to 1000 nm. In some embodiments, the liposomenanoparticles can have a size of from 10 to 150 nm.

In certain embodiments, the liposome nanoparticles of this invention canencapsulate the RNAi molecule and retain at least 80% of theencapsulated RNAi molecules after 1 hour exposure to human serum.

Pharmaceutical Compositions

This invention further contemplates methods for distributing an activeagent to an organ of a subject for treating malignant tumor byadministering to the subject a composition of this invention. Organsthat can be treated include lung, liver, pancreas, colon, heart, bone,skin, intestine and joints.

In some embodiments, this invention provides methods for treating a lungmalignant tumor disease by administering to the subject a composition ofthis invention.

In further aspects, this invention provides a range of pharmaceuticalformulations.

A pharmaceutical formulation herein can include an active agent, as wellas a drug carrier, or a lipid of this invention, along with apharmaceutically acceptable carrier or diluent. In general, activeagents of this description include siRNAs, active agents for malignanttumor, as well as any small molecule drug.

A drug carrier may target a composition to reach stellate cells. A drugcarrier may include a drug in its interior, or be attached to theexterior of a drug-containing substance, or be mixed with a drug so longas a retinoid derivative and/or vitamin A analogue is included in thedrug carrier, and is at least partially exposed on the exterior of thepreparation. The composition or preparation may be covered with anappropriate material, such as, for example, an enteric coating or amaterial that disintegrates over time, or may be incorporated into anappropriate drug release system.

A pharmaceutical formulation of this invention may contain one or moreof each of the following: a surface active agent, a diluent, anexcipient, a preservative, a stabilizer, a dye, and a suspension agent.

Some pharmaceutical carriers, diluents and components for apharmaceutical formulation, as well as methods for formulating andadministering the compounds and compositions of this invention aredescribed in Remington's Pharmaceutical Sciences, 18th Ed., MackPublishing Co., Easton, Pa. (1990).

Examples of preservatives include sodium benzoate, ascorbic acid, andesters of p-hydroxybenzoic acid.

Examples of surface active agents include alcohols, esters, sulfatedaliphatic alcohols.

Examples of excipients include sucrose, glucose, lactose, starch,crystallized cellulose, mannitol, light anhydrous silicate, magnesiumaluminate, magnesium metasilicate aluminate, synthetic aluminumsilicate, calcium carbonate, sodium acid carbonate, calcium hydrogenphosphate, and calcium carboxymethyl cellulose.

Examples of suspension agents include coconut oil, olive oil, sesameoil, peanut oil, soya, cellulose acetate phthalate,methylacetate-methacrylate copolymer, and ester phthalates.

A therapeutic formulation of this invention for the delivery of one ormore molecules active for gene silencing can be administered to a mammalin need thereof. A therapeutically effective amount of the formulationand active agent, which may be encapsulated in a liposome, can beadministered to a mammal for preventing or treating malignant tumor.

The route of administration may be local or systemic.

A therapeutically-effective formulation of this invention can beadministered by various routes, including intravenous, intraperitoneal,intramuscular, subcutaneous, and oral.

Routes of administration may include, for example, parenteral delivery,including intramuscular, subcutaneous, intravenous, intramedullaryinjections, as well as intrathecal, direct intraventricular,intraperitoneal, intranasal, or intraocular injections.

The formulation can also be administered in sustained or controlledrelease dosage forms, including depot injections, osmotic pumps, and thelike, for prolonged and/or timed, pulsed administration at apredetermined rate.

The composition of the present invention may be administered via variousroutes including both oral and parenteral routes, and examples thereofinclude, but are not limited to, oral, intravenous, intramuscular,subcutaneous, local, intrapulmonary, intra-airway, intratracheal,intrabronchial, nasal, rectal, intraarterial, intraportal,intraventricular, intramedullar, intra-lymph-node, intralymphatic,intrabrain, intrathecal, intracerebroventricular, transmucosal,percutaneous, intranasal, intraperitoneal, and intrauterine routes, andit may be formulated into a dosage form suitable for each administrationroute. Such a dosage form and formulation method may be selected asappropriate from any known dosage forms and methods. See e.g. HyojunYakuzaigaku, Standard Pharmaceutics, Ed. by Yoshiteru Watanabe et al.,Nankodo, 2003.

Examples of dosage forms suitable for oral administration include, butare not limited to, powder, granule, tablet, capsule, liquid,suspension, emulsion, gel, and syrup, and examples of the dosage formsuitable for parenteral administration include injections such as aninjectable solution, an injectable suspension, an injectable emulsion,and a ready-to-use injection. Formulations for parenteral administrationmay be a form such as an aqueous or nonaqueous isotonic sterile solutionor suspension.

Pharmaceutical formulations for parenteral administration, e.g., bybolus injection or continuous infusion, include aqueous solutions of theactive formulation in water-soluble form. Suspensions of the activecompounds may be prepared as appropriate oily injection suspensions.Aqueous injection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents that increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

Formulations for injection may be presented in unit dosage form, e.g.,in ampoules or in multi-dose containers, with an added preservative. Theformulations may take such forms as suspensions, solutions or emulsionsin oily or aqueous vehicles, and may contain formulary agents such assuspending, stabilizing and/or dispersing agents. Alternatively, theactive ingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

In addition to the preparations described previously, the formulationsmay also be formulated as a depot preparation. Such long actingformulations may be administered by intramuscular injection. Thus, forexample, the formulation may be formulated with suitable polymeric orhydrophobic materials, for example as an emulsion in an acceptable oil,or ion exchange resins, or as sparingly soluble derivatives, forexample, as a sparingly soluble salt.

Compositions and formulations of this invention may also be formulatedfor topical delivery and may be applied to the subject's skin using anysuitable process for application of topical delivery vehicle. Forexample, the formulation may be applied manually, using an applicator,or by a process that involves both. Following application, theformulation may be worked into the subject's skin, e.g., by rubbing.Application may be performed multiple times daily or on a once-dailybasis. For example, the formulation may be applied to a subject's skinonce a day, twice a day, or multiple times a day, or may be applied onceevery two days, once every three days, or about once every week, onceevery two weeks, or once every several weeks.

The formulations or pharmaceutical compositions described herein may beadministered to the subject by any suitable means. Examples of methodsof administration include, among others, (a) administration viainjection, subcutaneously, intraperitoneally, intravenously,intramuscularly, intradermally, intraorbitally, intracapsularly,intraspinally, intrasternally, or the like, including infusion pumpdelivery; (b) administration locally such as by injection directly inthe renal or cardiac area, e.g., by depot implantation; as well asdeemed appropriate by those of skill in the art for bringing the activecompound into contact with living tissue.

The exact formulation, route of administration and dosage for thepharmaceutical compositions can be chosen by the individual physician inview of the patient's condition. See, e.g., Goodman & Gilman's ThePharmacological Basis of Therapeutics, 12^(th) Ed., Sec. 1, 2011.Typically, the dose range of the composition administered to the patientcan be from about 0.5 to about 1000 mg/kg of the patient's body weight.The dosage may be a single one or a series of two or more given in thecourse of one or more days, as is needed by the patient. In instanceswhere human dosages for compounds have been established for at leastsome condition, the dosages will be about the same, or dosages that areabout 0.1% to about 500%, more preferably about 25% to about 250% of theestablished human dosage. Where no human dosage is established, as willbe the case for newly-discovered pharmaceutical compositions, a suitablehuman dosage can be inferred from ED50 or ID50 values, or otherappropriate values derived from in vitro or in vivo studies, asqualified by toxicity studies and efficacy studies in animals.

Methods for Preventing or Treating Malignant Tumor

The present invention further relates to a method for controlling theactivity or growth of malignant tumors, the method includingadministering an effective amount of the composition to a subject inneed thereof. The effective amount referred to here is, in a method fortreating malignant tumor, alleviates its symptoms, or delays or stopsits progression, and is preferably an amount that prevents the onset orrecurrence of malignant tumor, or cures it. It is also preferably anamount that does not cause an adverse effect that exceeds the benefitfrom administration. Such an amount may be determined as appropriate byan in vitro test using cultured cells or by a test in a model animal ormammal such as a mouse, a rat, a dog, or a pig, and such test methodsare well known to a person skilled in the art. Moreover, the dose of theactive agents in the carrier and the dose of the active agents used inthe method of the present invention are known to a person skilled in theart, or may be determined as appropriate by the above-mentioned tests.

The frequency of administration depends on the properties of thecomposition used and the above-mentioned conditions of the subject, andmay be a plurality of times per day (that is, 2, 3, 4, 5, or more timesper day), once a day, every few days (that is, every 2, 3, 4, 5, 6, or 7days, etc.), a few times per week (e.g. 2, 3, 4 times, etc. per week),every other week, or every few weeks (that is, every 2, 3, 4 weeks,etc.).

In some embodiments, the present invention also relates to a method fordelivering a drug to a malignant tumor cell, by utilizing the abovecarrier. This method includes a step of administering or adding thecarrier having the substance to be delivered carried thereon to a livingbeing or a medium, for example a culture medium, containing anextracellular matrix-producing cell in the lung. These steps may beachieved as appropriate in accordance with any known method or a methoddescribed in this invention. Moreover, the above method includes a modecarried out in vitro and a mode in which a malignant tumor cell in thelung inside the body is targeted.

A therapeutically-effective formulation of this invention can beadministered by systemic delivery that can provide a broadbiodistribution of the active agent.

Embodiments of this invention can provide a therapeutic formulation,which includes an inventive therapeutic molecule and apharmaceutically-acceptable carrier.

An effective dose of a formulation of this invention may be administeredfrom 1 to 12 times per day, or once per week. The duration ofadministration can be 1, 2, 3, 4, 5, 6 or 7 days, or can be 1, 2, 3, 4,5, 6, 8, 10 or 12 weeks.

EXAMPLES Example 1

An in vivo study was performed to test tumor growth inhibition withHSP47 as a single target. A pancreatic cancer model derived from PANC-1was chosen due to its enrichment of collagen proteins, as shown in Table5.

TABLE 5 In vivo study to test tumor growth inhibition with HSP47 as asingle target Animal model Dosing Group and # of Dosage Volume Dose IDanimals Treatment (mg/kg) (mL/kg) regimen Endpoint 1 PANC-1 PBS N/A 10mL/kg IV, BW daily within 2 7 animals Cmpd81:CH: 0.75 q1w × 4 7 dayspost first per group DOPE:DOPC: dosing; 2/week (~200 mm³) DPPE-PEG- forthe rest of 2K-2M week. Tumor measurement 3/for 1st week, 2/for the restof week. Terminal tumor weight

Study Methods:

PANC-1 cells were inoculated into right flank of female athymic nudemice subcutaneously.

Tumor size was measured and calculated using the formula: Tumorvolume=length×width/2. When the established tumors reached approximately200 mm³, the mice were randomized and assigned for test articleinjection.

Formulation containing HSP47-siRNA (2M) was injected into animalintravenously at 0.75 mg/kg, qlw and totals 4 doses.

Animal body weight and tumor volumes were monitored twice a week.

Results and Conclusion: Tumors grew slowly in this PANC-1 model, withdoubling time being 22 days for vehicle group. There was no body weightloss examined. The formulation, which was based on compound 81 andcontained the HSP47-siRNA (2M), was observed to completely suppresstumor growth at dosages of 0.75 mpk. Overall, suppression of Hsp47inhibited tumor growth, showing that the formulation was a potentanticancer therapeutic.

Results and Conclusion: FIG. 1 shows the results of an in vivo study ofa pancreatic cancer model that was performed to test tumor growthinhibition using Hsp47 as a single target. As shown in FIG. 1, for thegroup treated with a formulation containing Hsp47 siRNA (2M), the tumorvolumes grew slowly, if at all, during the course of the study. Therewas no body weight loss examined. To the contrary, the tumor volumes ofthe vehicle control group doubled in only 22 days. In conclusion, theHsp47 siRNA was observed to completely suppress tumor growth at dosagesof 0.75 mpk, showing that the formulation containing the Hsp47 siRNA wasa potent anticancer therapeutic.

Example 2

The gene expressions of Hsp47, collagen I, and collagen IV in humancancer cell lines A549, MCF7, MDA-MB-231, HCT116, M7609, COLO230HSR,SW480, PANC-1, SW1990, MIA-PaCa-2, HepG2, HT1080, and HaLa are shown inFIG. 2. Expressions are pronounced in SW480 and HepG2.

Example 3

Results of a method for suppressing proliferation of SW480 colon cancercells with a siRNA targeted to Hsp47 are shown in FIGS. 3-11.

FIG. 3 shows an untreated sample for a method for suppressingproliferation of SW480 colon cancer cells with a siRNA targeted toHsp47. FIG. 4 shows the effect of a negative siRNA at 10 nM in a methodfor suppressing proliferation of SW480 colon cancer cells with a siRNAtargeted to Hsp47. FIG. 5 shows the effect of a negative siRNA at 50 nMin a method for suppressing proliferation of SW480 colon cancer cellswith a siRNA targeted to Hsp47. FIG. 6 shows the effect of a negativesiRNA at 100 nM in a method for suppressing proliferation of SW480 coloncancer cells with a siRNA targeted to Hsp47. FIG. 7 shows the effect ofan active siRNA at 10 nM in a method for suppressing proliferation ofSW480 colon cancer cells with a siRNA targeted to Hsp47. FIG. 8 showsthe effect of an active siRNA at 50 nM in a method for suppressingproliferation of SW480 colon cancer cells with a siRNA targeted toHsp47. FIG. 9 shows the effect of an active siRNA at 100 nM in a methodfor suppressing proliferation of SW480 colon cancer cells with a siRNAtargeted to Hsp47.

Comparison of FIGS. 4 and 7, 5 and 8, and 6 and 9 shows that the activesiRNA targeted to Hsp47 suppresses SW480 colon cancer cells.

FIG. 10 shows the results of a growth assay in a method for suppressingproliferation of SW480 colon cancer cells with a siRNA targeted toHsp47. The vertical axis is the number of cells×10⁴. Filled circles arethe untreated sample. X markers are the negative siRNA. Triangle markersrepresent the sample treated with an active siRNA targeted to Hsp47.

FIG. 11 shows the results of a dye exclusion assay in a method forsuppressing proliferation of SW480 colon cancer cells with a siRNAtargeted to Hsp47. The vertical axis is the percentage of dead cells.Open circles are the sample treated with an active siRNA targeted toHsp47. X markers are the negative siRNA. Filled circle markers representthe untreated sample.

Example 4

Results of a method for suppressing proliferation of HCT116 colon cancercells with a siRNA targeted to Hsp47 are shown in FIGS. 12-16.

FIG. 12 shows an untreated sample in a method for suppressingproliferation of HCT116 colon cancer cells with a siRNA targeted toHsp47. FIG. 13 shows the effect of a negative siRNA at 10 nM in a methodfor suppressing proliferation of HCT116 colon cancer cells with a siRNAtargeted to Hsp47. FIG. 14 shows the effect of an active siRNA at 10 nMin a method for suppressing proliferation of HCT116 colon cancer cellswith a siRNA targeted to Hsp47. Comparison of FIGS. 12, 13 and 14 showsthat the active siRNA targeted to Hsp47 suppresses HCT116 colon cancercells.

FIG. 15 shows the results of a growth assay in a method for suppressingproliferation of HCT116 colon cancer cells with a siRNA targeted toHsp47. The vertical axis is the number of cells×10⁴. Filled circles arethe untreated sample. Open circle markers in the rising curve are thenegative siRNA. Open circle markers in the flat curve represent thesample treated with an active siRNA targeted to Hsp47.

FIG. 16 shows the results of a dye exclusion assay in a method forsuppressing proliferation of HCT116 colon cancer cells with a siRNAtargeted to Hsp47. The vertical axis is the percentage of dead cells.Open circles in the rising curve are the sample treated with an activesiRNA targeted to Hsp47. Open circle markers in the flat curve are thenegative siRNA. Filled circle markers represent the untreated sample.

Example 5

Results of a method for suppressing proliferation of A549 lung cancercells with a siRNA targeted to Hsp47 are shown in FIGS. 17-25.

FIG. 17 shows an untreated sample in a method for suppressingproliferation of A549 lung cancer cells with a siRNA targeted to Hsp47.FIG. 18 shows the effect of a negative siRNA at 10 nM in a method forsuppressing proliferation of A549 lung cancer cells with a siRNAtargeted to Hsp47. FIG. 19 shows the effect of a negative siRNA at 50 nMin a method for suppressing proliferation of A549 lung cancer cells witha siRNA targeted to Hsp47. FIG. 20 shows the effect of a negative siRNAat 100 nM in a method for suppressing proliferation of A549 lung cancercells with a siRNA targeted to Hsp47. FIG. 21 shows the effect of anactive siRNA at 10 nM in a method for suppressing proliferation of A549lung cancer cells with a siRNA targeted to Hsp47. FIG. 22 shows theeffect of an active siRNA at 50 nM in a method for suppressingproliferation of A549 lung cancer cells with a siRNA targeted to Hsp47.FIG. 23 shows the effect of an active siRNA at 100 nM in a method forsuppressing proliferation of SW480 colon cancer cells with a siRNAtargeted to Hsp47.

Comparison of FIGS. 18 and 21, 19 and 22, and 20 and 23 shows that theactive siRNA targeted to Hsp47 suppresses A549 lung cancer cells.

FIG. 24 shows the results of a growth assay in a method for suppressingproliferation of A549 lung cancer cells with a siRNA targeted to Hsp47.The vertical axis is the number of cells×10⁴. Filled circles are theuntreated sample. Open circle markers are the negative siRNA. Trianglemarkers represent the sample treated with an active siRNA targeted toHsp47.

FIG. 25 shows the results of a dye exclusion assay in a method forsuppressing proliferation of A549 lung cancer cells with a siRNAtargeted to Hsp47. The vertical axis is the percentage of dead cells.Open circles are the sample treated with an active siRNA targeted toHsp47. X markers are the negative siRNA. Filled circle markers representthe untreated sample.

Example 6

Results of a method for suppressing proliferation of A549 lung cancercells with a siRNA targeted to Hsp47 are shown in FIGS. 26-34.

FIG. 26 shows an untreated sample in a method for suppressingproliferation of HepG2 hepatic cancer cells with a siRNA targeted toHsp47. FIG. 27 shows the effect of a negative siRNA at 10 nM in a methodfor suppressing proliferation of HepG2 hepatic cancer cells with a siRNAtargeted to Hsp47. FIG. 28 shows the effect of a negative siRNA at 50 nMin a method for suppressing proliferation of HepG2 hepatic cancer cellswith a siRNA targeted to Hsp47. FIG. 29 shows the effect of a negativesiRNA at 100 nM in a method for suppressing proliferation of HepG2hepatic cancer cells with a siRNA targeted to Hsp47. FIG. 30 shows theeffect of an active siRNA at 10 nM in a method for suppressingproliferation of HepG2 hepatic cancer cells with a siRNA targeted toHsp47. FIG. 31 shows the effect of an active siRNA at 50 nM in a methodfor suppressing proliferation of HepG2 hepatic cancer cells with a siRNAtargeted to Hsp47. FIG. 32 shows the effect of an active siRNA at 100 nMin a method for suppressing proliferation of HepG2 hepatic cancer cellswith a siRNA targeted to Hsp47.

Comparison of FIGS. 27 and 30, 28 and 31, and 29 and 32 shows that theactive siRNA targeted to Hsp47 suppresses HepG2 hepatic cancer cells.

FIG. 33 shows the results of a growth assay in a method for suppressingproliferation of HepG2 hepatic cancer cells with a siRNA targeted toHsp47. The vertical axis is the number of cells×10⁴. Filled circles arethe untreated sample. Open circle markers are the negative siRNA. Xmarkers represent the sample treated with an active siRNA targeted toHsp47.

FIG. 34 shows the results of a dye exclusion assay in a method forsuppressing proliferation of HepG2 hepatic cancer cells with a siRNAtargeted to Hsp47. The vertical axis is the percentage of dead cells.Open circles are the sample treated with an active siRNA targeted toHsp47. X markers are the negative siRNA. Open square markers representthe untreated sample.

Example 7

Results of a method for detecting annexin V and PI in colon cancer cells(SW480, HCT116) transfected with an Hsp47 siRNA are shown in FIGS.35-36.

FIG. 35 shows the results of a method for detecting annexin V and PI incolon cancer cells SW480 transfected with an active Hsp47 siRNA on day 2after transfection of the siRNA. FIG. 36 shows the results of a methodfor detecting annexin V and PI in colon cancer cells HCT116 transfectedwith an active Hsp47 siRNA on day 2 after transfection of the siRNA.

Example 8

Results of a method for detecting expression of procaspase-3 and Hsp47protein in cancer cells transfected with an Hsp47 siRNA are shown inFIGS. 37-39.

FIG. 37 shows the results of a method for detecting expression ofprocaspase-3 and Hsp47 protein in SW480 colon cancer cells transfectedwith an active Hsp47 siRNA. FIG. 38 shows the results of a method fordetecting expression of procaspase-3 and Hsp47 protein in HepG2 hepaticcancer cells transfected with an active Hsp47 siRNA. FIG. 39 shows theresults of a method for detecting expression of procaspase-3 and Hsp47protein in A549 lung cancer cells transfected with an active Hsp47siRNA.

Example 9

Results of a method for detecting Caspase-3/7 activity in colon cancercells (SW480) transfected with an Hsp47 siRNA and a p21 siRNA are shownin FIG. 40. These data show that the use of a combination of an Hsp47siRNA and a p21 siRNA in colon cancer cells provided unexpectedlyadvantageous increases in the levels of Caspases. The surprisingincreases in the level of Caspases in cancer cells shows that the cellshave surprisingly increased apoptosis.

Example 10

In vitro transfection was performed in an A549 cell line to determinesiRNA knockdown efficacy. Using a formulation of this inventioncontaining RNAi molecules, which are targeted to Hsp47, dose dependentknockdown for Hsp47 mRNA is observed.

Protocol for in vitro knockdown: One day before the transfection, platethe cells in a 96-well plate at 2×103 cells per well with 100 μl of DMEM(HyClone Cat. # SH30243.01) containing 10% FBS and culture in a 37° C.incubator containing a humidified atmosphere of 5% CO2 in air. Beforetransfection, change medium to 90 μl of Opti-MEM I Reduced Serum Medium(Life Technologies Cat. #31985-070) containing 2% FBS. Mix 0.2 μl ofLipofectamine RNAiMax (Life Technologies Cat. #13778-100) with 4.8 μl ofOpti-MEM I for 5 minutes at room temperature. Mix 1 μl of siRNA with 4μl of Opti-MEM I and combine with the LF2000 solution and then mixgently, without vortex. Wait for 5 minutes at room temperature. Incubatethe mixture for 10 minutes at room temperature to allow the RNA-RNAiMaxcomplexes to form. Add the 10 μl of RNA-RNAiMax complexes to a well andshake the plate gently by hand. Incubate the cells in a 37° C. incubatorcontaining a humidified atmosphere of 5% CO2 in air for 2 hours. Changemedium to fresh-MEM I Reduced Serum Medium (Life Technologies Cat.#31985-070) containing 2% FBS. 24 hours after transfection, wash thecells with ice-cold PBS once. Lyse the cells with 50 μl of Cell-to-CtLysis Buffer (Life Technologies Cat. #4391851 C) for 5-30 minutes atroom temperature. Add 5 μl of Stop Solution and incubate for 2 minutesat room temperature. Measure mRNA level by RT-qPCR with TAQMANimmediately. Alternatively, the samples can be frozen at −80° C. andassayed at a later time.

Example 11

Tumor inhibition efficacy for Hsp47 siRNA. A pancreatic cancer xenograftmodel is utilized with a relatively low dose at 0.75 mg/kg of siRNAtargeted to Hsp47. The combined siRNAs demonstrate significant andunexpectedly advantageous tumor inhibition efficacy at day 28.

In this experiment, A549 and PANC-1 cell lines are obtained from ATCC.The cell suspension is mixed well with ice thawed BD matrigel at 1:1ratio for injection. Each mouse, athymic nude female mice, 6 to 8 weeks,Charles River, is inoculated subcutaneously in the right flank with 0.1ml of an inoculum of 2×10⁶ (A549) or 2.5×10⁶ (PANC-1) cells using a 25 Gneedle and syringe (1 inoculum per mouse). Mice are anesthetized forinoculation. On the day when the established tumors reachesapproximately 250-350 mm³ (A549) or 150-250 mm³ (PANC-1) animals aresubjected to bolus injection through tail vein. Animals are sacrificedby overdosed CO₂ and tumors dissected at different time points followingthe dosing. Tumors are first wet weighted, and then are separated intothree parts for measurement of Hsp47 knockdown, biodistribution ofsiRNA, and biomarker analysis. The samples are snap frozen in liquidnitrogen and stored at −80° C. until ready to be processed forbioanalysis.

Example 12

Efficacy evaluation of siRNA encapsulated in a liposomal formulation onan orthotopic A549 lung cancer mouse model.

Experimental Animals: A total of sixty male NCr nu/nu mice, 5-6 weeksold, are used in the study. The experimental animals are bred and raisedat AntiCancer Inc. They are maintained in a HEPA filtered environmentduring the experimental period. Cages, food and bedding are autoclaved.The animal diets are obtained from Harlan Teklad (Madison, Wis.).

Liposomal formulation preparation: The formulations are prepared andstored at 4° C. They are warmed to room temperature 10 minutes prior toinjecting to the mice.

A549 human lung cancer orthotopic model (SOI): On the day of SOI, thestock tumors are harvested from the subcutaneous site of animals bearingA549 tumor xenograft and placed in RPMI-1640 medium. Necrotic tissuesare removed and viable tissues are cut into 1.5-2 mm³ pieces. Theanimals are anesthetized with isoflurane inhalation and the surgicalarea is sterilized with iodine and alcohol. A transverse incisionapproximately 1.5 cm long is made in the left chest wall of the mouseusing a pair of surgical scissors. An intercostal incision is madebetween the third and the fourth rib and the left lung is exposed. OneA549 tumor fragment is transplanted to the surface of the lung with an8-0 surgical suture (nylon). The chest wall is closed with a 6-0surgical suture (silk). The lung is re-inflated by intrathoracicpuncture using a 3 cc syringe with a 25 G×1½ needle to draw out theremaining air in the chest cavity. The chest wall is closed with a 6-0surgical silk suture. All procedures of the operation described aboveare performed with a 7× magnification microscope (Olympus) under HEPAfiltered laminar flow hoods.

Three days after tumor implantation, the tumor-bearing mice are randomlydivided into groups with ten mice per group. Treatments for each groupof mice are initiated three days after tumor implantation.

Endpoint: The experimental mice are sacrificed forty-two days aftertreatment initiation. Primary tumors are excised and weighed on anelectronic balance for subsequent analysis.

The anti-tumor efficacy of formulations against human lung cancer A549is evaluated by comparing the final primary tumor weights measured atnecropsy between each of the treatment groups and the vehicle controlgroup. The average tumor weight in each group is measured.

Estimation of compound toxicity: The mean body weight of the mice in thetreated and control groups is maintained within the normal range duringthe entire experimental period. Other symptoms of toxicity are also notdetected by gross observation of the mice.

CONCLUSION: By comparing the final tumor weights that are obtained atthe end of the experiment, it is concluded that treatment with theformulation containing the Hsp47 siRNA at 2 mg/kg in the treatmentgroups significantly and surprisingly reduces tumor growth and tumorvolume of the human lung cancer A549 by comparison with controls.Toxicity is not observed.

Example 13

Effect of small interfering RNA (siRNA) targeting Hsp47 on A549 cellgrowth in nude mice and angiogenesis on chorioallantoic membrane (CAM)assay. Three pairs of Hsp47 siRNA-plasmid and non-silencing-plasmid areconstructed, and transfected into A549 cells through LIPOFECTAMINE 2000,respectively. The most effective pair of Hsp47 siRNA-plasmid is selectedby ELISA and real-time RT-PCR. A549 cells are transfected with selectedHsp47 siRNA-plasmid, A549 cells are transfected withnon-silencing-plasmid, and A549 cells without transfection areinoculated into nude mice, respectively. Chick embryos are randomlydivided into four groups and CAM is treated by different solutions for48 h: culture media DMEM as negative control group, un-transfected A549cell culture supernatants as positive control group, Hsp47 siRNA A549cell culture supernatants as Hsp47 siRNA group and non-silencing siRNAA549 cell culture supernatants as non-silencing siRNA group. The CAMswere harvested on day 12 for microscopic assays.

Compared with control group, Hsp47 siRNA-plasmid induces reduction inHsp47 secretion by A549 cells accompanied by reduction in Hsp47 mRNA.Compared with non-silencing siRNA group, the mean tumor volume of murinexenograft is reduced in Hsp47 siRNA group; time for xenografts growingto 50 mm³ is delayed. Hsp47 contents in xenograft are reduced. In CAMassays, Hsp47 content is zero in negative group, and in Hsp47 siRNAgroup is reduced by 20-70% compared to non-silencing siRNA group orpositive group; vessels branch points of CAM in Hsp47 siRNA group ornon-silencing siRNA group or positive group are increased compared withnegative group; total vessel length of CAM in Hsp47 siRNA group isincreased compared with negative group, while in non-silencing siRNAgroup or positive group it is increased. Compared with negative controlgroup, the proliferation of microvessels is increased when cell culturesupernatant with Hsp47 added in Hsp47 siRNA group, significantproliferated vessels are observed in non-silencing siRNA group orpositive group.

Example 14

Cell culture. The human non-small cell lung carcinoma cell line, A549 iscultured in F-12K medium (ATCC) supplemented with 10% FBS (FBS,Invitrogen) at 37° C. in a humidified atmosphere with 5% CO₂. The cellsstably expressing control, orHsp47 siRNAs are generated by transducingA549TR cells with the respective lentiviral transduction particles asper manufacturer's instructions (Sigma-Aldrich). Resistant clones areselected in 2.5 μg/mL puromycin (Invivogen) for 12 d, isolated usingcloning cylinders, and subsequently expanded and maintained inpuromycin-containing medium.

Example 15

Hsp47 targeted siRNAs result in profound regression of tumor volume invivo.

A lipid formulation is used to encapsulate and deliver siRNA innanoparticles to xenografts of human A549 lung cancer cells in scidmice. The xenografts are tested to identify the presence of KRASmutations or aberrant levels of expression compared to normal cells.When tumors became established (>100 mm³), mice are treated with eitherHsp47 targeted siRNA or Control (non-specific) siRNA every 2 days for 2weeks. The trial is halted when the control group has to be euthanized.

Results: Treatment with Hsp47 targeted siRNA prevents tumor expansionand results in dramatic tumor volume reduction.

The tumors that are recovered are sectioned and visualized by TUNELstaining. Hsp47 targeted siRNA-treated tumors display significantlyhigher levels of apoptosis. RNA is extracted from the tumors, andreal-time PCR is performed to examine specific knockdown of Hsp47.

Results: Treatment with Hsp47 targeted siRNA dramatically reducesexpression of Hsp47 in vivo.

Example 16

siRNAs of this invention targeted to p21 were found to be active forgene silencing in vitro. The dose-dependent activities of p21 siRNAs forgene knockdown were found to exhibit an IC50 below about 3 picomolar(pM), and as low as 1 pM.

In vitro transfection was performed in an A549 cell line to determinesiRNA knockdown efficacy. Dose dependent knockdown for p21 mRNA wasobserved with siRNAs of Table 1, as shown in Table 6.

TABLE 6 Dose dependent knockdown for p21 mRNA in an A549 cell line P21siRNA structure IC50 (pM) 1735 (SEQ ID NOs: 12 and 40) 0.3 2042 (SEQ IDNOs: 28 and 56) 10

As shown in Table 6, the activities of p21 siRNAs of Table 1 were in therange 0.3-10 pM, which is suitable for many uses, including as a drugagent to be used in vivo.

Example 17

The structure of p21 siRNAs of this invention having deoxynucleotideslocated in the seed region of the antisense strand of the siRNA providedunexpectedly and advantageously increased gene knockdown activity.

In vitro transfection was performed in an A549 cell line to determineknockdown efficacy for p21 siRNAs based on structure 1735′ (SEQ IDNOs:57 and 71). Dose dependent knockdown of p21 mRNA was observed withp21 siRNAs based on structure 1735′ as shown in Table 7.

TABLE 7 Dose dependent knockdown of p21 mRNA in an A549 cell line forp21 siRNAs based on structure 1735′ P21 siRNA structure IC50 (pM) 1735with no deoxynucleotides in the duplex region 0.3 (SEQ ID NOs: 12 and40) 1735 with deoxynucleotides in positions 4, 6, and 8 0.05 of the seedregion antisense strand, and additional 2′-OMe nucleotides (SEQ ID NOs:58 and 72) 1735 with deoxynucleotides in positions 4, 6, and 8 0.001 ofthe seed region antisense strand, and additional 2′-OMe nucleotides (SEQID NOs: 59 and 73) 1735 with deoxynucleotides in positions 4, 6, and 80.1 of the seed region antisense strand, and additional 2′-OMenucleotides (SEQ ID NOs: 60 and 74)

As shown in Table 7, the activities of p21 siRNAs based on structure1735′ having three deoxynucleotides in the seed region of the antisensestrand were surprisingly and unexpectedly increased by up to 300-fold,as compared to a p21 siRNA without deoxynucleotides in the duplexregion.

These data show that p21 siRNAs having a structure with deoxynucleotidesin the seed region of the antisense strand provided surprisinglyincreased gene knockdown activity as compared to a p21 siRNA withoutdeoxynucleotides in the duplex region.

The activities shown in Table 7 for p21 siRNAs having threedeoxynucleotides in the seed region of the antisense strand were in therange 0.001 to 0.1 pM, which is exceptionally suitable for many uses,including as a drug agent to be used in vivo.

The embodiments described herein are not limiting and one skilled in theart can readily appreciate that specific combinations of themodifications described herein can be tested without undueexperimentation toward identifying nucleic acid molecules with improvedRNAi activity.

All publications, patents and literature specifically mentioned hereinare incorporated by reference in their entirety for all purposes.

It is understood that this invention is not limited to the particularmethodology, protocols, materials, and reagents described, as these mayvary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention. It will be readilyapparent to one skilled in the art that varying substitutions andmodifications can be made to the description disclosed herein withoutdeparting from the scope and spirit of the description, and that thoseembodiments are within the scope of this description and the appendedclaims.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise. As well, the terms “a” (or “an”),“one or more” and “at least one” can be used interchangeably herein. Itis also to be noted that the terms “comprises,” “comprising”,“containing,” “including”, and “having” can be used interchangeably, andshall be read expansively and without limitation.

Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein. For Markush groups, those skilled in theart will recognize that this description includes the individualmembers, as well as subgroups of the members of the Markush group.

Without further elaboration, it is believed that one skilled in the artcan, based on the above description, utilize the present invention toits fullest extent. The following specific embodiments are, therefore,to be construed as merely illustrative, and not limitative of theremainder of the disclosure in any way whatsoever.

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose.

What is claimed is:
 1. A pharmaceutical composition for treatingmalignant tumor, the composition comprising nanoparticles encapsulatingRNAi molecules, wherein the RNAi molecules are targeted to Hsp47.
 2. Thepharmaceutical composition of claim 1, wherein the composition retainsat least 80% of the activity of the encapsulated RNAi molecules after 1hour exposure to human serum.
 3. The pharmaceutical composition of claim1, wherein the RNAi molecules for treating malignant tumor are siRNAs orshRNAs.
 4. The pharmaceutical composition of claim 1, wherein each RNAimolecule comprises a duplex region, wherein the duplex region comprisesa nucleotide sequence corresponding to a target sequence of Hsp47 mRNA.5. A method for distributing an active agent to a subject for treatingmalignant tumor, the method comprising administering to the subject apharmaceutical composition of claim
 1. 6. A pharmaceutical compositionfor treating malignant tumor, the composition comprising nanoparticlesencapsulating RNAi molecules, wherein a portion of the RNAi moleculesare targeted to Hsp47 and a portion of the RNAi molecules are targetedto p21.
 7. The pharmaceutical composition of claim 6, wherein thecomposition retains at least 80% of the activity of the encapsulatedRNAi molecules after 1 hour exposure to human serum.
 8. Thepharmaceutical composition of claim 6, wherein the RNAi molecules fortreating malignant tumor are siRNAs or shRNAs.
 9. The pharmaceuticalcomposition of claim 6, wherein for the portion of the RNAi moleculesthat are active in reducing expression of Hsp47, each RNAi moleculecomprises a duplex region, wherein the duplex region comprises anucleotide sequence corresponding to a target sequence of Hsp47 mRNA.10. The pharmaceutical composition of claim 6, wherein for the portionof the RNAi molecules that are active in reducing expression of p21,each RNAi molecule comprises a duplex region, wherein the duplex regioncomprises a nucleotide sequence corresponding to a target sequence ofp21 mRNA.
 11. A method for distributing an active agent to a subject fortreating malignant tumor, the method comprising administering to thesubject a pharmaceutical composition of claim
 6. 12. The method of claim11, wherein the malignant tumor is located in the lung, colon, orpancreas, liver, heart, bone, skin, or intestine.
 13. The method ofclaim 11, wherein the administration is a dose of from 0.01 to 2 mg/kgof the RNAi molecules at least once per day for a period up to twelveweeks.
 14. The method of claim 11, wherein the administration provides amean AUC (0-last) of from 1 to 1000 ug*min/mL and a mean C_(max) of from0.1 to 50 ug/mL for the Hsp47 RNAi molecule.
 15. A method forpreventing, treating or ameliorating one or more symptoms of a malignanttumor in a mammal in need thereof, the method comprising administeringto the mammal a therapeutically effective amount of a compositioncomprising RNAi molecules active in reducing expression of Hsp47. 16.The method of claim 15, wherein the mammal is a human, the Hsp47 is ahuman Hsp47.
 17. The method of claim 15, wherein the malignant tumoroverexpresses Hsp47.
 18. The method of claim 15, wherein the RNAimolecules decrease expression of Hsp47 in the mammal.
 19. The method ofclaim 15, wherein the administration decreases expression of Hsp47 inthe mammal by at least 5% for at least 5 days.
 20. The method of claim15, wherein the administration decreases the volume of the malignanttumor in the mammal by at least 5%, or at least 10%, or at least 20%, orat least 30%, or at least 40%, or at least 50%.
 21. The method of claim15, wherein the method reduces one or more symptoms of the malignanttumor, or delays or terminates the progression of the malignant tumor.22. The method of claim 15, wherein the administration reduces growth ofmalignant tumor cells in the subject.
 23. The method of claim 15,wherein the administration reduces growth for at least 2%, or at least5%, or at least 10%, or at least 15%, or at least 20% of the malignanttumor cells in the subject.
 24. The method of claim 15, wherein thetumor cells overexpress wild-type Hsp47 RNA or protein.
 25. The methodof claim 15, wherein the tumor is a sarcoma selected from the groupconsisting of lung adenocarcinoma, mucinous adenoma, ductal carcinoma ofthe pancreas and colorectal carcinoma.
 26. The method of claim 15,wherein the malignant tumor is a sarcoma selected from the group of lungadenocarcinoma, mucinous adenoma, ductal carcinoma of the pancreas,colorectal carcinoma, breast cancer, and fibrosarcoma.
 27. The method ofclaim 15, wherein the malignant tumor is located in an anatomical regionselected from the group of lung, liver, pancreas, colon, kidney, heart,bone, skin, intestine and joints, and any combination thereof.
 28. Themethod of claim 15, wherein the administration is performed from 1 to 12times per day.
 29. The method of claim 15, wherein the administration isperformed for a duration of 1, 2, 3, 4, 5, 6 or 7 days.
 30. The methodof claim 15, wherein the administration is performed for a duration of1, 2, 3, 4, 5, 6, 8, 10 or 12 weeks.
 31. The method of claim 15, whereinthe administration is a dose of from 0.01 to 2 mg/kg of the RNAimolecules at least once per day for a period up to twelve weeks.
 32. Themethod of claim 15, wherein the administration provides a mean AUC(0-last) of from 1 to 1000 ug*min/mL and a mean C_(max) of from 0.1 to50 ug/mL for the Hsp47 RNAi molecule.
 33. The method of claim 15,wherein the administration is intravenous injection, intradermalinjection, subcutaneous injection, intramuscular injection,intraperitoneal injection, oral, topical, infusion, or inhaled.
 34. Amethod for preventing, treating or ameliorating one or more symptoms ofa malignant tumor in a mammal in need thereof, the method comprisingadministering to the mammal a therapeutically effective amount of acomposition comprising RNAi molecules, wherein a portion of the RNAimolecules are active in reducing expression of Hsp47 and a portion ofthe RNAi molecules are active in reducing expression of p21.
 35. Themethod of claim 34, wherein the mammal is a human, the Hsp47 is a humanHsp47.
 36. The method of claim 34, wherein the malignant tumoroverexpresses Hsp47.
 37. The method of claim 34, wherein the RNAimolecules decrease expression of Hsp47 and p21 in the mammal.
 38. Themethod of claim 34, wherein the administration decreases expression ofHsp47 and p21 in the mammal by at least 5% for at least 5 days.
 39. Themethod of claim 34, wherein the administration decreases the volume ofthe malignant tumor in the mammal by at least 5%, or at least 10%, or atleast 20%, or at least 30%, or at least 40%, or at least 50%.
 40. Themethod of claim 34, wherein the method reduces one or more symptoms ofthe malignant tumor, or delays or terminates the progression of themalignant tumor.
 41. The method of claim 34, wherein the administrationreduces growth of malignant tumor cells in the subject.
 42. The methodof claim 34, wherein the administration reduces growth for at least 2%,or at least 5%, or at least 10%, or at least 15%, or at least 20% of themalignant tumor cells in the subject.
 43. The method of claim 34,wherein the tumor cells overexpress wild-type Hsp47 RNA or protein. 44.The method of claim 34, wherein the tumor is a sarcoma selected from thegroup consisting of lung adenocarcinoma, mucinous adenoma, ductalcarcinoma of the pancreas and colorectal carcinoma.
 45. The method ofclaim 34, wherein the malignant tumor is a sarcoma selected from thegroup of lung adenocarcinoma, mucinous adenoma, ductal carcinoma of thepancreas, colorectal carcinoma, breast cancer, and fibrosarcoma.
 46. Themethod of claim 34, wherein the malignant tumor is located in ananatomical region selected from the group of lung, liver, pancreas,colon, kidney, heart, bone, skin, intestine and joints, and anycombination thereof.
 47. The method of claim 34, wherein theadministration is performed from 1 to 12 times per day.
 48. The methodof claim 34, wherein the administration is performed for a duration of1, 2, 3, 4, 5, 6 or 7 days.
 49. The method of claim 34, wherein theadministration is performed for a duration of 1, 2, 3, 4, 5, 6, 8, 10 or12 weeks.
 50. The method of claim 34, wherein the administration is adose of from 0.01 to 2 mg/kg of the RNAi molecules at least once per dayfor a period up to twelve weeks.
 51. The method of claim 34, wherein theadministration provides a mean AUC (0-last) of from 1 to 1000 ug*min/mLand a mean C_(max) of from 0.1 to 50 ug/mL for the Hsp47 RNAi molecule.52. The method of claim 34, wherein the administration is intravenousinjection, intradermal injection, subcutaneous injection, intramuscularinjection, intraperitoneal injection, oral, topical, infusion, orinhaled.
 53. A method for reducing the growth rate or proliferation ofcancer stem cells in a mammal in need thereof, the method comprisingadministering to the mammal a therapeutically effective amount of acomposition comprising RNAi molecules, wherein a portion of the RNAimolecules are active in reducing expression of Hsp47 and a portion ofthe RNAi molecules are active in reducing expression of p21.
 54. Themethod of claim 53, wherein the mammal is a human, the Hsp47 is a humanHsp47.
 55. The method of claim 53, wherein the RNAi molecules decreaseexpression of Hsp47 and p21 in the mammal.
 56. The method of claim 53,wherein the administration decreases expression of Hsp47 and p21 in themammal by at least 5% for at least 5 days.
 57. The method of claim 53,wherein the administration decreases the volume of the malignant tumorin the mammal by at least 5%, or at least 10%, or at least 20%, or atleast 30%, or at least 40%, or at least 50%.
 58. The method of claim 53,wherein the administration reduces growth for at least 2%, or at least5%, or at least 10%, or at least 15%, or at least 20% of cancer stemcells in the subject.
 59. The method of claim 53, wherein the cancercells overexpress wild-type Hsp47 RNA or protein.
 60. The method ofclaim 53, wherein the cancer cells are in a sarcoma selected from thegroup consisting of lung adenocarcinoma, mucinous adenoma, ductalcarcinoma of the pancreas and colorectal carcinoma.
 61. The method ofclaim 53, wherein the cancer cells are in a sarcoma selected from thegroup of lung adenocarcinoma, mucinous adenoma, ductal carcinoma of thepancreas, colorectal carcinoma, breast cancer, and fibrosarcoma.
 62. Themethod of claim 53, wherein the cancer cells are located in ananatomical region selected from the group of lung, liver, pancreas,colon, kidney, heart, bone marrow, skin, intestine, eye, and anycombination thereof.
 63. The method of claim 53, wherein theadministration is performed from 1 to 12 times per day.
 64. The methodof claim 53, wherein the administration is performed for a duration of1, 2, 3, 4, 5, 6 or 7 days.
 65. The method of claim 53, wherein theadministration is performed for a duration of 1, 2, 3, 4, 5, 6, 8, 10 or12 weeks.
 66. The method of claim 53, wherein the administration is adose of from 0.01 to 2 mg/kg of the RNAi molecules at least once per dayfor a period up to twelve weeks.
 67. The method of claim 53, wherein theadministration provides a mean AUC (0-last) of from 1 to 1000 ug*min/mLand a mean C_(max) of from 0.1 to 50 ug/mL for the Hsp47 RNAi molecule.68. The method of claim 53, wherein the administration is intravenousinjection, intradermal injection, subcutaneous injection, intramuscularinjection, intraperitoneal injection, oral, topical, infusion, orinhaled.
 69. A composition for use in treating a malignant tumor in asubject, wherein the composition comprises liposome nanoparticles thatencapsulate RNAi molecules targeted to Hsp47.
 70. The composition ofclaim 69, wherein the malignant tumor is located in the lung, colon, orpancreas.
 71. The composition of claim 69, wherein the malignant tumoris located in the liver, heart, bone, skin, or intestine.
 72. Thecomposition of claim 69, wherein the composition comprises liposomenanoparticles having a size of from 10 to 1000 nm.
 73. The compositionof claim 69, wherein the composition comprises liposome nanoparticleshaving a size of from 10 to 150 nm.
 74. The composition of claim 69,wherein a portion of the RNAi molecules are targeted to Hsp47 and aportion of the RNAi molecules are targeted to targeted to p21.
 75. Thecomposition of claim 74, wherein the liposome nanoparticles retain atleast 80% of the encapsulated RNAi molecules after 1 hour exposure tohuman serum.
 76. A method for distributing an active agent to an organof a subject for treating malignant tumor, the method comprisingadministering to the subject a composition of claim
 69. 77. The methodof claim 76, wherein the malignant tumor is located in the lung, colon,kidney, pancreas, liver, bone marrow, skin, eye or intestine.